bluuebunny/crossref_metadata_2025 huggingface dataset:
Beneficial Effects of the Melaleuca Alternifolia Essential Oil in the Treatment of Diabetic Ulcers.
Authors: González-Palomares Lizbeth , Gómez-Barroso Mariana , Vargas-Vargas Manuel Alejandro , Cortés-Rojo Christian , Saavedra-Molina Alfredo , Salgado-Garciglia Rafael , Pérez Rocío Montoya-
Published: April 2020

Currently, the incidence of diabetes mellitus (DM) in the world population has obtained epidemic proportions, becoming one of the leading causes of death. One of the main complications of this disease are ulcers caused by neuropathy and diabetic vasculopathy. Diabetic ulcers can be prevented with proper follow‐up. However, pharmacological treatments for such pathology are usually expensive, lengthy, and inefficient. Ethereal oils or essential oils are a complex mixture, mainly of secondary metabolites of lipophilic and highly volatile plants, they are highly concentrated. Tea tree essential oil is obtained from the leaves and ailerons of Melaleuca alternifolia. Its main constituent is terpinen‐4‐ol. This compound is considered to be primarily responsible, together with other components, such as α‐terpinene, γ‐terpinene, and α‐terpineol, for the antibacterial, antifungal, antiprotozoic, anti‐inflammatory and antioxidant activities of the oil. The objective of this work was to evaluate the effect of Melaleuca alternifolia oil on the healing of diabetic ulcers and the deterioration of muscle strength caused by them. Male Wistar rats (250 g) were used in four groups: wounded diabetic rats treated with melaleuca alternifolia, healthy wounded rats treated with melaleuca alternifolia, diabetic rats wounded without treatment, healthy rats wounded without treatment. The long extensor finger muscle (EDL) of one of the extremities and the biceps femoral muscle of the other limb were injured by simulating diabetic ulcers. These wounds were treated with essential oil of Melaleuca alternifolia for 11 days. During the administration of the treatment, the injury and its closing progress were monitored, measuring their diameter. At the end of the procedure, the EDL muscle was removed for muscle tension tests. The results showed that diabetic wounded rats treated with Melaleuca and untreated closed simultaneously at eleven days. However, untreated wounded rats remained open until the ninth day, showing a diameter of 7 mm at the beginning of treatment and a diameter of 4 mm on the ninth day, while the rats injured with Melaleuca began to close after the third day, showing a diameter of 7 mm at the beginning of the treatment and a diameter of 4 mm on the third day and progressing until closing. On the other hand, the results of muscle tension showed that diabetic wounded rats treated with Melaleuca improved both maximum tension by 22% and total tension by 26% compared to the untreated injured diabetic group. In conclusion, it was observed that the essential oil of Melaleuca alternifolia has a significant beneficial effect on healing and cell proliferation, as well as muscle function.
Inhibition of Sphingosine Kinase 2 Attenuated Cisplatin‐induced Nephrotoxicity in Mice
Authors: Xie Dengpiao Li , Hu Gaizun , Wang Weili , Li Pin-Lan , Chen Chaoling , Li Ningjun
Published: April 2020

Sphingosine‐1‐phosphate (S1P) is a bioactive sphingolipid metabolite and shown to participate in various kidney diseases. S1P is produced by sphingosine kinases (Sphks), which phosphorylate sphingosine into S1P. It has been demonstrated that knockout of Sphk2 gene protects the kidneys in unilateral ureteral obstruction model. The present study tested the hypothesis that inhibition of Sphk2 protects the kidneys against cisplatin‐induced nephrotoxicity. Male C57BL6 mice were divided into three groups: vehicle (Ctrl), cisplatin + vehicle (Cis) and cisplatin + ABC294640, a Sphk2 inhibitor (Cis + in). Cisplatin (10 mg/Kg) was given by weekly i.p. injection and Sphk2 inhibitor (35 mg/Kg) gavage every other day for 4 weeks. Plasma creatinine levels were significantly higher in Cis group than that in Ctrl, whereas it was significantly lower in Cis+in group than that in Cis (mean: 0.17 ± 0.02, 0.38 ± 0.05, and 0.20 ± 0.02 mg/dL in Ctrl, Cis and Cis+in group, respectively, p<0.05). Western blot analysis showed that the levels of fibrotic protein alpha‐smooth muscle actin (α‐SMA) in the kidneys were significantly increased in Cis group and that this increase in α‐SMA was blocked in Cis+in group. The relative α‐SMA protein levels were 1.0 ± 0.04, 1.8 ± 0.11 and 0.8 ± 0.19 in Ctrl, Cis and Cis+in group, respectively (p<0.05). The increased level of Kidney Injury Molecule‐1 was also significantly reduced in Cis+in group compared with that in Cis group. These results suggest that Sphk2 pathway mediates cisplatin‐induced kidney damage. Manipulating Sphk2 may be used as a therapeutic strategy in the management of cisplatin‐induced nephrotoxicity.

Support or Funding Information

NIH grants R01DK54927, R01DK107991, R01HL145163
ADAM17 impairs arterial fluid shear stress mechanosensing in aged mice
Authors: Tian Yanna , Davila Alec , Clermont Andrew , Balogh Marta Bagine , Czikora Istvan , Bagi Zsolt
Published: April 2020

Junctional Adhesion Molecule A (F11R/JAM‐A), which can be shed by a disintegrin and metalloproteinase ADAM17, has been implicated in arterial mechanotransduction. We hypothesized that in aging ADAM17 activation impairs wall shear stress mechanosensing via an augmented F11R/JAM‐A shedding. Using videomicroscopy we found a reduced wall shear stress (WSS)‐induced dilation in isolated, pressurized gracilis muscle arteries in 30‐month old (aged) mice, when compared to 12‐week old (young) controls. Dilations to acetylcholine or to the NO donor, sodium nitroprusside were not different between the two groups. We found that ADAM17 expression was significantly upregulated in arteries in aged mice, which was associated with reduced endothelial cell junction localized F11R/JAM‐A. After activation of ADAM17 with phorbol‐myristat‐acetate, WSS‐induced dilation was reduced in young mice, whereas ADAM17 inhibition by TIMP3 augmented WSS‐induced dilation in aged mice. Cultured HUVECs exposed to increasing levels of WSS (0 to 16 dyne/cm2) progressively changed their orientation to the direction of flow. In the presence of recombinant ADAM17 or after siRNA‐mediated silencing of F11R/JAM‐A, endothelial cells failed to efficiently orient with the direction of flow. Gene transfer of ADAM17 cleavage resistant, mutated JAM‐A(V227Y) resulted in a normal endothelial orientation to flow even after ADAM17 activation. Collectively, our results uncover a novel role for ADAM17 in impairing F11R/JAM‐A‐dependent WSS mechanosensing in arteries of aged mice.

Support or Funding Information

National Institute of Aging (R01AG054651)
Loss of CLC‐5 reduces endocytic capacity and alters cholesterol distribution in the proximal tubule
Authors: Shipman Katherine , Long Kimberly R. , Rbaibi Youssef , Raja Sripriya , Baty Catherine J. , Weisz Ora A.
Published: April 2020

Dent disease is a progressive X‐linked disorder affecting the kidney proximal tubule (PT) caused by loss of function of the Cl−/H+ exchanger, CLC‐5. Early symptoms include low molecular weight (LMW) proteinuria resulting from inefficient reabsorption of filtered proteins by the PT. However, the role of CLC‐5 in this process in unclear. Knockout of Clc‐5 in mice recapitulates the LMW proteinuria observed in human disease and causes decreased PT levels of megalin and cubilin, the receptors that bind to and recover filtered proteins. How loss of CLC‐5 leads to reduced receptor expression remains unknown. We used a previously developed opossum kidney (OK) cell culture model that recapitulates morphologic and functional features of the PT in vivo to study the role of Clc‐5 in the endocytic pathway. In Clc‐5 siRNA knockdown (KD) and CRISPR/Cas9 mediated Clc‐5 knockout (KO) cells, there was a significant decrease in endocytic capacity that was rescued by heterologous expression of wild‐type human CLC‐5. Additionally, we used CRISPR/Cas9 technology to generate Clc‐5 knockout mice and confirmed LMW proteinuria in the KO. Heterozygous females also have reduced PT albumin uptake and megalin expression. Previous gene expression studies in Clcn5 KO mice suggest there are alterations in cholesterol and lipid metabolic enzymes, and elevated cholesterol levels have been shown to alter recycling endosome organization and impair fast recycling. We hypothesize that loss of CLC‐5 disrupts cholesterol homeostasis, resulting in reduced recycling and concomitantly increased degradation of megalin. Consistent with this, we observed an accumulation and redistribution of cholesterol in Clc‐5 KD OK cells. We are currently working to quantify cholesterol in Clc‐5 KO OK cells. In female mice with only one functional copy of Clc‐5, we observed a dramatic a change in the distribution of cholesterol in the PT when compared to WT mice. Current studies are focused on quantifying the trafficking kinetics and distribution of megalin in the PT in the absence of CLC‐5 and determining whether altered cholesterol metabolism contributes to the reduced endocytic capacity of the PT and resulting LMW proteinuria in Dent disease.

Support or Funding Information

NIH F31 DK121394, R01 DK101484, R01 DK100357, P30 DK079307, and ASN Pre‐Doctoral Fellowship
Cytotoxic activity of proteins extracted from fennel (Foeniculum vulgare) seeds against human cancer cell lines
Authors: Megeressa Mekdes , Do Van , Ahmed Aftab
Published: April 2020

Fennel (Foeniculum vulgare) is an aromatic flowering medicinal plant classified into the family Umbelliferae (Apiaceae). It is native to southern Europe and the Mediterranean region with a long history of use by humans as a spice, fresh vegetable, and in traditional folk medicine. Despite its popularity for its medicinal value, the majority of data in scientific literature is based on the crude extracts and essential oils such as trans‐anethole and estragole, fenchone, limonene, and 1‐octen‐3‐ol. Besides, other phytochemicals found from seeds are polyphenols, included rosmarinic acid and luteolin, etc. In present study, we investigated the anticancer potential of proteins purified from fennel seeds against human breast cancer MCF 7 and pancreatic cancer AsPC‐1 cell lines. The crude fennel extract was partially purified using anion‐exchange and size exclusion chromatography techniques to have a comparative analysis for activity guided fractionation. For anion‐exchange chromatography (HiTrap‐Q FF 5ml column in 20mM Tris/HCl, pH 8.00 buffer) and for gel filtration (HiPrep Sephacryl S‐200 HR16/20 column in 20mM, pH 8.00 buffer ) were used. The crude extract and fractions collected were judged for their purity by 10% Tris‐tricine SDS‐PAGE gel electrophoresis. The MTT assay results exhibited that both anion‐exchange and gel filtration fractions demonstrated significant cytotoxic activity against MCF‐7 breast cancer cell line with cell inhibition in a range of 65–80% at 100μg/ml. In contrast, evaluation againist AsPC‐1pancreatic cancer cell line revealed non‐significant inhibition by fractions obtained from both methods. Further work is in progress to identify and fully characterized the primary structure of cytotoxic proteins by mass spectrometry and Edman protein sequencing.
Implementing Evidence‐Based Teaching Practices: Support for Educators through the Promoting Active Learning & Mentoring Research Coordination Network (PALM Network)
Authors: Johnson Kathryn M.S. , Stieben Margaret E. , Wick Sue
Published: April 2020

The Promoting Active Learning & Mentoring Research Coordination Network (PALM Network) provides one‐to‐one year‐long mentored support to biology faculty and postdoctoral fellows seeking to implement evidence‐based, effective active learning strategies into their classrooms. These PALM fellows are paired with mentors, recognized for their experience in education reform, typically faculty at another academic institution. Mentors assist fellows as they design, implement, and assess teaching innovations in their own classroom, and fellows visit the mentor’s institution to observe evidence‐based practices in the mentor’s classroom. In addition to individual mentoring, PALM fellows meet regularly, virtually and in‐person, with other fellows and mentors, providing a diverse and broad network of support for their continued development as educators. Support and assistance from professional societies provides an operational framework, promotion, and recognition to the Network. To date, 28 fellow‐mentor pairs have participated in the PALM Network. Here we present the findings of the mid‐point evaluation of the 5‐year NSF‐funded network, using a mixed‐methods assessment approach. Fellows (n=13) and mentors (n=11) completed online surveys upon acceptance or after one year of participation in the PALM Network. At the time of acceptance into the PALM Network, self‐reported confidence of the network member’s ability to use backward design to develop active learning activities and assessments of student learning was dependent upon whether the respondent was a fellow or a mentor (p ≤ 0.046). For those surveyed after one year in the PALM Network, self‐reported confidence in these abilities were not different between fellows and mentors (p ≥ 0.122), indicating the abilities of the fellows shifted towards that of the mentors. Grounded‐theory analysis of semi‐structured in‐person interviews with mentors and fellows (n=8, representing 7 mentor‐fellow pairs) participating in the PALM Network for at least 6 months, but less than 2 years, revealed PALM Network members are enthusiastic, talented, and ambitious educators and researchers, dedicated to implementing education reform. PALM fellows value the one‐to‐one mentoring and the support of a broad network. PALM Network fellow‐mentor collaborations result in successful implementation of transformational pedagogical projects. Although implementing change at the fellow’s home institution was sometimes difficult, the mentors and other PALM Network members provided valuable advice and support for the fellows to facilitate their work. Both fellows and mentors highly recommend the PALM Network. Therefore, the PALM Network provides effective support for educators implementing evidence‐based best practices in their classrooms.

Support or Funding Information

This work was funded by NSF‐RCN‐UBE #1624200, including a sub‐award to American Physiological Society.
Integrating Innovative Handheld Portable Cordless Ultrasound‐Probe‐System with Artificial Intelligence for Pre‐Healthcare Students in Human Cadaver Lab Course
Authors: Rivas-Vasquez Luis , Estrada Ruben , Benninger Brion
Published: April 2020

INTRODUCTION

The importance of ultrasound regarding its depth and breadth across medical fields continues to expand in development and use while affordability improves for the user and patient. Sonoanatomy is important for interpreting the anatomical architecture adjacent to the probe. Medical educators responsible for teaching ultrasound are faced with two obstacles, teaching how to drive the ultrasound technology and understanding the surface, static and dynamic sonoanatomy of the regions of the body.

AIM

The aim of this study was to identify and teach sonoanatomy with ultrasound which limits or removes driving the knobology associated with traditional ultrasound technology.

METHODS

Literature search was conducted to identify an ultrasound probe system which is handheld, cordless, with no buttons and uses artificial intelligence to provide diagnostic quality imaging. An upper division baccalaureate semester cadaver dissection course (pre healthcare Gen Z students N=14) which included sonoanatomy teaching and examination with a traditional cart ultrasound probe system was the arena to introduce such technology. Students rotated during every dissection lab to practice ultrasound probe positioning, image acquisition and anatomy interpretation.

RESULTS

Clarius handheld wireless multiprobe ultrasound‐probe‐system compatible with Apple and Android mobile devices was identified and used equally with traditional cart ultrasound. Students were successfully taught surface and regional sonoanatomy in lectures and tutorials using exam results.

DISCUSSION

Clarius is shock and water resistant and utilizes automated AI with a real time automated time‐gain‐compensation (TGC) engine. Traditional ultrasound systems require users adjusting several TGC controls (knobs) to optimize imaging and often when changing scanning planes, attenuation variance forces users to adjust TGC often resulting in failed images adding to the learning curve. Clarius automated AI overrides user interaction with TGC controls if desired. Analyzing each single ultrasound image captured during scanning at rates of 30 frames per second, gain can be tuned with high level precision to 1mm, all in real‐time, with instant feedback resulting in quality images.

CONCLUSION

Students preferred not having to adjust knobs and performed better acquiring quality images with Clarius probe system on cadaver and simulation exercises then with a traditional ultrasound cart system. This pilot project suggests more Clarius ultrasound studies could be used to train novices in healthcare.
Sex Differences in Sepsis Survival Involve Effects of Gonadal Steroid Hormones
Authors: Chakraborty Sanjukta , Venkatasamy Lavanya , Stallone John N.
Published: April 2020

Sepsis is one of the leading causes of death in human non‐coronary induced intensive care cases; in the United States, more than 750,000 people are diagnosed with sepsis each year, and 20 to 30% of those die from severe sepsis. While advances in aggressive supportive care have resulted in a reduction in the number of deaths from sepsis each year, the total number of cases of sepsis continues to increase. Given the complexity and severity of sepsis, more successful treatment will require thorough elucidation of the various inflammatory mechanisms. Thus, in the present study, sex differences in sepsis survival and the role(s) of the gonadal steroid hormones were studied. Age‐matched male (M) and female (F) Sprague‐Dawley rats (12–14 wks age) either remained intact (InT) or were gonadectomized (GnX) using standard surgical methods. Four weeks later, sepsis was induced in InT (N = 7) and GnX (N = 3) M and F rats using standard cecal ligation and puncture (CLP) methods while under isoflurane anesthesia (50% of cecum ligated, single bilateral puncture, 20 ga hypodermic needle). Immediately after CLP, rats were resuscitated with 40 ml/kg warmed (37°) saline, given a single dose of buprenorphine SR (1.0 mg/kg), and mortality was monitored over the following 10 days. A separate group of InT M and F rats underwent sham CLP surgery. Kaplan‐Meier analysis of the data revealed that survival was 100% in both M and F sham groups. In InT CLP groups, by day 3, there was a drastic reduction in survival to 42.8% in M but 57.1% in F; this sex difference increased over time and by day 6, maximal survival was 14.3% in M but 42.8% in F. In GnX, at day 3, survival declined to 33% in M, but increased to 100% in F. These data suggest that prominent sex differences exist in survival from CLP‐induced sepsis, which may involve effects of the gonadal steroid hormones on inflammatory and immune function in M vs. F rats.

Support or Funding Information

(State of Texas)
SPARC: A Carbon Fiber Nerve Electrode Appropriate for Chronic Recording
Authors: Welle Elissa Joy , Jiman Ahmad A. , Patel Paras R. , Woods Joshua E. , Richie Julianna M. , Bottorff Elizabeth C. , Seymour John P. , Bruns Tim M. , Chestek Cynthia A.
Published: April 2020

State‐of‐the‐art intraneural peripheral nerve electrodes are large, silicon‐based structures that can cause substantial tissue response and are ill‐suited for recording from small autonomic nerves. The focus of this work is to adapt our minimally‐scarring carbon fiber brain electrodes to a chronic intraneural nerve array. The objective of this study is to increase the durability of the carbon fiber electrodes to withstand the surgical handling necessary for nerve implantation, while maintaining a cellular scale electrode that can insert through the outer epineurium layer of peripheral nerves. Toward that end, we embedded carbon fibers in silicone to increase robustness, sharpened the fibers to penetrate epineurium, and tested fibers coated with poly(3,4‐ethylene‐dioxythiophene):sodium p‐toluenesulfonate (PEDOT:pTS) in vivo.

We tested the durability of carbon fibers by embedding uninsulated fibers in a body of silicone and inducing a 90‐degree bend to simulate the expected shear forces seen during surgery. We found that fibers of 175μm length were robust to thousands of bend cycles. After 3000 bends, 15 of 16 fibers remained intact. We blunt cut carbon fibers using a 532nm green laser and inserted fibers into silicone of stiffness similar to epineurium to determine a length for in vivo testing. We found that blunt fibers reliably insert into silicone without breaking when less than 200μm in length (N=8 fibers). We hypothesized that sharpened fibers would insert into tissue more easily, and therefore sharpened fibers with a 5mm butane flame when protruding from the surface of water. The dimensions of sharpened fibers were 224μm in length with 147μm of exposed carbon and a tip angle of 72 degrees (N=24 fibers).

We inserted functionalized sharpened carbon fibers coated with PEDOT:pTS into peroneal and cervical vagus nerves of anesthetized rats (N=5 arrays, 71 fibers, 7 animals). In all experiments, we observed that the sharpened carbon fiber electrodes penetrated the epineurium for successful insertion. The average 1kHz impedance of coated fibers prior to and upon insertion was 38kΩ and 56kΩ, respectively. In both cases, the Z1kHz of roughly 90% of fibers measured below 100kΩ. We recorded spontaneous and evoked neural activity in each experiment and are analyzing the data for discernable single units. We also inserted sharpened fibers into anesthetized feline sacral dorsal root ganglia (N=3) and confirmed insertion with evoked neural recordings and 1kHz impedances (Z=59kΩ).

This work shows that the durable nature of carbon fibers embedded in silicone allows them to withstand surgical handling and insert into various types of nerves. Our next step will be the fabrication of an array for chronic implantation.

Support or Funding Information

NIH OT2OD024907, NINDS U01NS094375, NINDS UF1NS107659, NSF 1707316
Skin Temperature Response to a 1 km Swim Performed in Normal and Cold‐Water
Authors: Lentz Mason , Fell Megan , Ahlers Kristina , Clementson Samantha , Frie Kathryn , Johnson Bruce D. , Harrison Michael F. , Wilson Ted
Published: April 2020

Cold‐water swimming is a recreational activity that may become a future Winter Olympic event. However, unintentional cold‐water exposure can lead to hypothermia or death. Changes in body temperature are known to diminish swimming performance in persons experiencing hypothermia, although thermal changes during recreational exposure are not well characterized. This study examined skin temperature responses to a one‐kilometer swim in normal and cold‐water. Competitive swimmers (2 male and 2 female; age 44±17 years; BMI 24±2) completed a one‐kilometer swim (22 laps × 75 foot lengths) under free‐living indoor (25.5°C water and 25.0°C air) and cold outdoor (10.9°C water and 6.5°C) conditions. Skin temperature was determined with a digital infrared camera on the center of right palm, cubital fossa of arm, lateral aspect of pectoral muscle, cheek, and average of the four locations at 8.7±1.7 minutes before the swim (pre), then 0.6±0.2 and 6.4±2.1 minutes after the swim. Data (mean±SD) was analyzed with repeated measures ANOVA with significance if P<0.05. Swimmers completed the indoor and outdoor swim events in 20.7±1.7 and 21.8±1.1 minutes respectively. Average indoor skin temperature at −8.7, +0.6 and +6.4 minutes was 94.0±1.7, 85.3±1.9, and 88.1±2.2°C. Average outdoor skin temperature at −8.7, +0.6 and +6.4 minutes was 83.0±3.6, 53.5±2.3, and 67.7±2.0°C. At all times average outdoor skin temperature was lower than indoor, with the lowest temperature 0.6 minutes post‐swim. Palm indoor temperature at −8.7, +0.6 and +6.4 minutes was 91.6±3.9, 83.1±0.5, and 87.3±5.0°C. Palm outdoor temperature at −8.7, +0.6 and +6.4 minutes was 75.1±7.9, 47.4±2.7, and 58.1±2.7°C. At all times outdoor palm temperature was lower than indoor, with the lowest temperature 0.6 minutes post‐swim. Similar thermal responses were observed in the cheek, cubital fossa and pectoral locations. While time to complete the indoor and outdoor one km swims were similar, swimming in cold water was associated with delayed rewarming in the palm and cheek relative to cubital fossa and pectoral muscle.
Identifying Prospective Genes That Function During DNA Repair in the Bdelloid Rotifer Adineta vaga
Authors: Wiles James Tristian , Schurko Andrew
Published: April 2020

The bdelloid rotifer Adineta vaga is a microinvertebrate with a highly efficient DNA repair system that allows individuals to recover from DNA damage caused by desiccation or ionizing radiation. The mechanism behind this remarkable DNA repair in A. vaga is not yet understood. Previous transcriptome sequencing in our lab has identified over 250 genes that are upregulated in response to DNA damage induced by ionizing radiation. These upregulated genes are the strongest candidates for having a role in DNA repair. The objective of this project is to characterize the functions of candidate genes involved in DNA repair in bdelloids and, thereby, establish potential pathways that comprise this mechanism. Three approaches were used to begin to investigate the functions of these candidate DNA repair genes in bdelloids. First, we used real‐time PCR to quantify gene expression of several candidate DNA repair genes during recovery from desiccation. Genes that were upregulated relative to the non‐desiccated control were identified as most likely to have a role in DNA repair as opposed to radiation resistance. Second, we constructed a yeast library of A. vaga proteins for use in yeast two‐hybrid screens against candidate DNA repair proteins. To produce the library, cDNAs representing the full complement of A. vaga proteins were cloned into plasmids that were then transformed into yeast. Yeast strains that each expressed a different candidate DNA repair protein would then be mated with the library to screen for protein‐protein interactions, which would provide evidence for related cellular functions. Third, we developed the molecular tools for using CRISPR/Cas9 genome editing to inactivate candidate genes in A. vaga. In vivo gene knockouts would provide mutant rotifers that could be studied to assess the roles of these genes in DNA repair. Single guide RNAs (sgRNAs) for several genes were created and incubated with the Cas9 enzyme to form sgRNA‐Cas9 complexes. These sgRNA‐Cas9 complexes were shown to successfully cleave their target gene sequences in vitro. To test this method in vivo, these sgRNA‐Cas9 complexes were transfected into A. vaga embryos via electroporation. Genomic DNA was purified from treated rotifers and used in T7 endonuclease I assays to identify subsequent mutations in the target genes. Once perfected, these methods of investigating the roles of candidate genes stand to elucidate the mechanism behind the extraordinary DNA repair in A. vaga.

Support or Funding Information

Funding was provided by a Summer Undergraduate Research Fellowship from the Arkansas Department of Higher Education to JTW, and a Hendrix College Faculty Project Grant to AMS.
Plastinated Human Samples Are Efficient Tools In Anatomy Teaching
Authors: Fang Xiaoqian
Published: April 2020

Most of the medical schools in the United States teach human anatomy using cadavers and/or collections of human organs and tissues stored in preservatives. Cadaveric dissections engage students in learning anatomy with real human samples and also provide them an opportunity to use their hands managing surgical tools and some surgical procedures. However, cadaveric dissection could be time‐consuming and may conflict with the current tendency of reducing contact hours in medical school curricula, particularly in gross anatomy teaching. Maintaining cadavers, organs and tissues require special space and additional time of management. Computer aided 3‐D technology as a complementary element to facilitate anatomy learning brings students a wealth of data in a short period of time. Especially, the deepest structures can be easily shown on computers (Apps). However, one major concern from students is that they are not able to recognize these structures in human bodies because “they look different to Atlas/Apps”. In this study, we aim to provide a novel approach in teaching gross anatomy. At UTRGV SOM, we equipped our anatomy lab with plastinated human samples. Plastination is a preservation technique in which polymers are used in substituting formaldehyde to fix tissues. Plastinates are dry, long‐lasting, and odorless. Students save time studying plastinates as opposed to working on cadaver dissections. In addition, exposures to possible toxic chemicals is limited to a low level while working with plastinates. Furthermore, plastinates could accurately reveal structures and structural relationships in comparison with 3‐D technology. However, due to the restriction of the techniques, plastinates are rigid and do not allow learners to investigate hidden structures and several plastinates with different preparations are required to serve the need of the demonstration from different aspects. In conclusion, from our experience, plastinates could be a novel approach to replace traditional cadaver dissection in teaching first‐ and second‐year medical students’ human anatomy. Using plastinates, our students benefit from real human sample experience and efficient content learning.
Phylogenetic Study of Beta‐Galactosidase in Model Organisms Using Bioinformatics and Western Blot Techniques
Authors: Wood Lance Phelan
Published: April 2020

Beta‐galactosidase is a highly conserved enzyme found in many organisms, existing as a tetramer of four identical subunits of 1023 amino acids each; its substrate is lactose, although it can bind to other similar molecules as well (Jeurs, D et. Al). It can cleave the lactose molecule to form allolactose, which is involved in the lac operon of E. coli, and glucose, which is used in glycolysis in many other organisms (Jeurs, D. et al). This experiment uses published protein sequences for the Beta‐galactosidase enzyme in four different model organisms to construct a phylogenetic tree using E. coli, S. cerevisiae, D. melanogaster, and C. elegans proteomes. Using Western Blot techniques, the same enzyme from all four species will be used to create a phylogenetic tree based on the experimental data. Beta Galactosidase Antibody (NBP2‐42819) is a polyclonal rabbit antibody from Novus Biologicals will be used as the primary antibody for binding to the extracted enzyme. Rabbit IgG Horseradish Peroxidase conjugated Antibody, catalog number HAF008, also from Novus Biologicals will be used as the secondary antibody to bind to the primary. The in‐silico model will then be compared with the in‐vitro model to compare antibody binding to the bacterial, fungal, and animal forms of the enzyme. Preliminary bioinformatics work based on multiple sequence alignments and domain searches suggests that C. elegans and D. melanogaster forms of beta‐galactosidase are more closely related to each other than to any of the forms found in the other species in this study. The S. cerevisiae form of the protein was expected to be more like those belonging to C. elegans and D. melanogaster. However, the bioinformatics results do not support this, possibly due to poor literary documentation of the amino acid sequence of S. cerevisiae beta‐galactosidase.

Support or Funding Information

This work was made possible through the contributions of the Hillsborough Community College Brandon Campus Biotechnology and Microbiology Departments.
Identifying Protein Targets of KDAC4 and KDAC5
Authors: Nguyen Trang T. , Phillips Tiffany , Toro Tasha B. , Watt Terry J.
Published: April 2020

Lysine acetylation is a common post‐translational modification that regulates many biological processes and that has been linked to numerous diseases. Protein lysine deacetylases (KDACs or HDACs) are enzymes that reverse this post‐translational modification by removing the acetyl group from the lysine residue in proteins. KDACs therefore play a critical role in the overall regulation of acetylation. Our objective is to understand the biological roles of KDAC4 and KDAC5, which are class IIa metal‐dependent KDACs, with an emphasis on identifying potential non‐histone substrates. Purified class IIa KDACs have greatly reduced catalytic activity in vitro compared to other KDACs, and only a few putative substrates have been identified for these enzymes in cell‐based experiments. To determine the possible targets of KDAC4 and KDAC5, we have recombinantly expressed and purified various enzyme constructs, including variants with site‐specific mutations that enhance activity to levels comparable with other metal‐dependent KDACs. Activity is measured using a combination of fluorescence‐based assays and mass spectrometry, using biologically relevant putative substrates. Activity assays using model peptides derived from known acetylated proteins with these gain‐of‐function variants reveal specific interaction preferences for the KDACs. Comparison of activity of the isolated catalytic domain with the activity of the full‐length proteins, which contain a second domain of unknown function, allows for determination of the impact of the second domain on interactions with possible target proteins. Together, our results provide insight into possible cellular targets of KDAC4 and KDAC5, as well as providing a mechanism to evaluate whether these enzymes are likely to be acting as deacetylases in vivo or interact with target proteins in a non‐catalytic manner. Understanding of the activity of these KDACs may lead to new therapeutic approaches for acetylation‐associated diseases.

Support or Funding Information

NIH TL4GM118968, UL1GM118967, 5G12MD007595, and R15GM129682; NSF CHE 1625993 and MCB 1817358; U. S. Army Research Laboratory and the U. S. Army Research Office W911NF1810450; and the Louisiana Cancer Research Consortium.
The Effect of Delta‐9‐Tetrahydrocannabinol and Cannabidiol on Blood Flow and Blood Pressure
Authors: James Tameika , Pepple Dagogo , Watson Charah
Published: April 2020

The effect of different concentrations of Delta‐9‐Tetrahydrocannabinol (THC) and Cannabidiol (CBD) separately and in combination on blood flow and blood pressure were investigated. It is important to fully elucidate the possible impact on the health of individuals who utilize cannabis and its products because cannabis has been increasingly implicated in adverse cardiovascular events.

Forty male Sprague‐Dawley rats were randomly divided into eight groups consisting of five rats each. One group served as a control group. Six of the test groups received different concentrations of THC and CBD (1, 2 and 4 mg/kg body weight) respectively, while the other test group received a combination of THC and CBD (2 mg/kg body weight) in a one to one ratio. The control group received vehicle only. Drugs were administered intraperitoneally on alternate days for five days. Blood flow and blood pressure were measured over a period of one hour prior to and after administration of THC and CBD using the CODA non‐invasive blood pressure system.

The results indicate that blood flow increases with increasing CBD concentration but decreases with an increase in THC concentration. These changes were however only significantly different from control at 45 mins when 2 mg/kg of either drug was administered.

The extract caused a decrease in blood flow which was not significantly different from control.

CBD had no significant effect on blood pressure. THC however caused a downward trend in blood pressure which was not significant. The extract caused a decrease in blood pressure which was significant at 30, 45 & 60 minutes.

The changes observed with increasing concentrations of THC and CBD suggest that these cannabinoids operate in dose dependent manner and that THC and CBD may have different effects on the same parameter when administered separately, but when used in combination one may modulate the effect of the other.

Support or Funding Information

Graduate Studies & Research, University of the West Indies, Mona Campus, Kingston, Jamaica
Influence of Acute Fasting on Cerebrovascular Reactivity During Mental Stress
Authors: Gonzalez Joshua Eric , Stelly Steven Philip , Cooke William Harold
Published: April 2020

Acute fasting has been adopted in fitness communities, and has been practiced for religious purposes. Short‐term fasting does not affect cognitive function, and therefore may not affect cerebrovascular responsiveness. Cerebral blood flow velocity increases with mental stress, however the combined influences of an acute fast and mental stress on cerebrovascular responses are unknown. The purpose of this study was to describe the effects of a 24‐hour, water‐only fast on cerebral blood flow velocity responses to mental stress in a small cohort of research volunteers. Eight young healthy participants (7 males, average age of 25 yrs) completed the protocol in a fed state (4 hours post‐prandial) and in a fasted state (24–25 hours post‐prandial). Whole blood was collected and analyzed before experimentation for β‐Ketones, total cholesterol, HDL, LDL, triglycerides (TRG), and glucose. With participants in a supine position, we recorded the electrocardiogram, beat‐to‐beat arterial pressure (finger plethysmography), and cerebral artery blood flow velocity (transcranial Doppler). After a 5‐min baseline, participants performed 5‐min of mental stress via mental arithmetic (rapid continuous subtraction). Mental stress reactivity was calculated as the mean stress response for each minute of mental stress minus the corresponding 5‐min baseline. The minute‐by‐minute reactivity was analyzed with repeated measures ANOVA. Blood variables were analyzed with paired t‐tests. Data were expressed as mean ± SE. Baseline blood variables were different in the fasted compared with the fed state: TRG (112±14 fed vs. 68±13 mg/dL fasted; p<.01) Glucose (101±5 fed vs. 79±6 mg/dL fasted; p<.01) and β‐Ketone (.1±.01 fed vs. .5±.2 mmol/L fasted; p<.05). Mean arterial pressures and heart rate reactivities to mental stress were not affected by fasting. Mean cerebral blood flow velocity reactivity to mental stress was reduced in the fasted compared with the fed condition (condition × time, p<.01), with a particular divergent responsiveness during the third minute of mental stress. The results we present are new, but preliminary. We did not attempt to ascribe mechanisms to fasting and cerebral blood flow velocity reactivity during mental stress. The changes we observed with our preliminary data set suggest that fasting, and associated changes of plasma glucose, β‐Ketone and TRG may be associated with reductions of cerebral blood flow velocity during mental stress.

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Support was provided by the Michigan Space Grant Consortium and The Portage Health Foundation of Michigan.
Potential mechanisms of opioid depression of the laryngeal adductor reflex: insights from a neuromechanical model of the respiratory control system
Authors: Olsen Wendy Lorraine , Pitts Teresa Greene , Morris Kendall F. , Segers Lauren S. , Shuman Dale , Nuding Sara C. , Bolser Donald C.
Published: April 2020

Opioid use increases the risk of aspiration and pneumonia, presumably through depression of protective reflexes that prevent intrusion of foreign material into the airways. The laryngeal adductor reflex (LAR) is an airway protective behavior that utilizes bilateral thyroarytenoid (TA) muscles and prevents material entering the trachea and lungs. The operational features and specific neural mechanisms which regulate and coordinate the occurrence of the LAR to prevent aspiration during opioid use are not fully understood. In vivo experiments were conducted using unilateral electrical stimulation of the superior laryngeal nerve (SLN) to induce the LAR. Systemic opioid administration preferentially attenuated the contralateral LAR, which is consistent with dysregulation of airway protection. We speculated that computational modeling of a specific LAR circuit would provide testable hypotheses that could account for this effect of opioids on the LAR. We have developed a predictive computational model which incorporates a distributed neural network that excites expiratory laryngeal motoneurons (ELM). The stochastic neural network simulator consists of discrete “integrate and fire” populations. The model circuit was informed by response patterns during the LAR of neurons in the regions of the nucleus tractus solitarius (NTS), the reticular formation (RF), and the ventrolateral respiratory column (VRC). With our network scale model, we simulated modulation of the LAR during administration of opioids by decreasing input currents to neurons in the RF that provide presynaptic input to ELMs of the VRC and then we activated laryngeal receptors. Simulator outputs were compared to collected in vivo data. Computer simulations reproduced the temporal features of the LAR, and the unilateral effects of opioids on the LAR by increasing potassium conductance of presynaptic neurons, depressing presynaptic terminal excitability, and hyperpolarizing ELM populations. Simulations of the LAR and phasic breathing suggest that an important area of focus for modeling efforts will be (i) refining the spatiotemporal characteristics of the behavior, (ii) the potential role of NTS laryngeal interneuronal networks in the effects of opioids, and (iii) synaptic regulation of oscillatory behavior of the LAR.

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Supported by NIH HL131716, 3OT2OD023854, and NS 110169.
In vitro Metabolism studies of Catechol‐Containing Natural Products and Analogues
Authors: Hermanson Jake , Bohannan Amanda , Schwartz Zachary , Jemal Mauricio , Kerwin Sean M.
Published: April 2020

Many biologically active natural products contain phenol groups that are prone to rapid phase‐II metabolism. In order to better understand the structural basis for these metabolic transformations, we have investigated the in vitro metabolism of rooperol, a bis‐catechol with anti‐cancer activity, and caffeic acid phenethyl amide (CAPA), a synthetic analog of a natural product with antioxidant and cytoprotective activities. In the presence of pig liver microsomes supplemented with UDP‐glucuronic acid, both of these catechols undergo rapid phase‐II metabolism to form mono‐ (CAPA and rooperol) and bis‐ (rooperol) glucuronide metabolites. The identity of these metabolites was determined by HPLC‐MS. We followed the kinetics of these in vitro metabolism reactions using HPLC. In both cases, the disappearance of the compound over time corresponded to a first‐order process with half‐life of 4.0 ± 0.5 min for rooperol and 5.1 ± 0.8 min for CAPA. In the case of rooperol, this data is in agreement with previously published pharmacokinetic (PK) studies in humans, in which only phase‐II metabolites were detected after oral administration, as well as PK studies in baboons which also report rapid phase‐II metabolism following IV administration. While human or non‐human primate PK data for CAPA have not been reported, our data suggest that this compound will also suffer from rapid phase‐II metabolism, and thus more metabolically stable analogs of both rooperol and CAPA are required.

Support or Funding Information

This research was supported by the U.S. Department of Education HSI STEM program (841.031c), Award #P021C160036

The Robert and Ella Owens Medical Research Foundation
Rapamycin‐Dependent Gln3 Transcription Factor Dephosphorylation and Nuclear Localization Depend On Distinct Regions of the Gln3 Protein
Authors: Tate Jennifer J. , Cooper Terrance G
Published: April 2020

Yeast cell survival in nitrogen‐depleted environments depends on Gln3 entering the nucleus and activating: (i) Nitrogen Catabolite Repression‐sensitive genes whose products scavenge poor nitrogen sources from its extra‐cellular surroundings and (ii) the ATG14 gene required for autophagic recycling nitrogen from intra‐cellular constituents. The TorC1 global serine/threonine kinase complex is one of multiple regulators that controls intracellular Gln3 localization and function. When TorC1 is specifically inhibited by rapamycin, Gln3 is dephosphorylated in a Sit4 phosphatase‐dependent manner, enters the nucleus and activates transcription. However, rapamycin‐elicited Gln3 dephosphorylation itself is insufficient to achieve this outcome, as the hypo‐phosphorylated Gln3 fails to enter the nucleus in the absence of a second phosphatase, PP2A. These observations led to the hypothesis that rapamycin‐elicited outcomes involve distinct target sequences within the Gln3 protein. Supporting this hypothesis, our experiments demonstrate that abolishing the Gln3‐Tor1 interaction, rapamycin‐response or Gln3 cytoplasmic sequestration targets abrogates nuclear Gln3 localization but without effect on rapamycin‐elicited Gln3 dephosphorylation. Truncated Gln3 proteins further show that summary loss of all three regulatory targets also has no effect on Gln3 dephosphorylation. Finally, we have identified multiple specific serine/threonine residues in the N‐terminal region of Gln3 that are dephosphorylated in rapamycin treated cells and when substituted with phosphomimetic aspartate residues abolish Gln3 localization in an otherwise full‐length wild type protein.

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This work supported by NIH GM35642‐27 and LTAUSA18162.
Different developmental transformations underly blowhole orientation in a toothed whale (Odontoceti: Stenella attenuata) and a baleen whale (Mysticeti: Balaenoptera physalus)
Authors: Roston Rachel A. , Roth V. Louise
Published: April 2020

Although the external bony nares have become posteriorly repositioned in the evolution of numerous groups of mammals (e.g. elephants, tapirs), reorientation of the nasal passage away from the anteroposterior axis has evolved rarely. In cetacean (whale, dolphin, porpoise) evolution, dorsoventral reorientation of the nasal passage along with extreme posteriad repositioning of the nares formed a “blowhole.” Despite the presence of a blowhole at birth, early cetacean fetuses exhibit head morphologies that resemble those of other mammals. Two distinct but non‐competing developmental models have been proposed for how the nasal passage reorients into a blowhole. In one model, which focused on external changes in the whole body, nasal passage reorientation was described in terms of dorsad rotation of the head during prenatal growth (Pilleri and Wandeler, 1962). A second, focusing on the nasal skull, noted that the nasal passage itself changes orientation relative to the palate and longitudinal axis of the skull (Klima, 1995, 1999). To integrate and revise these models, we used photos and CT scans of fetal growth series to characterize morphological, allometric, and angular changes within the head in representatives of the two extant cetacean suborders: a toothed whale, the pantropical spotted dolphin (Odontoceti: Stenella attenuata), and a baleen whale, the fin whale (Mysticeti: Balaenoptera physalus). In both fetal series, the angle between the nasal passage and the sagittal axis of the foramen magnum decreased with age, but this trend accompanied different morphological changes. In S. attenuata, the angle between the palate and basicranium flattened in association with facial lordosis and basicranial retroflexion in the region of the presphenoid. Basicranial retroflexion was not observed in B. physalus; rather, alignment of the nasal passage and the sagittal axis of foramen magnum accompanied anteriad tilting of the occipital bone. With these results, we propose a new developmental model, differing in its specifics for a toothed and a baleen whale, for the reorientations that produce the blowhole.