bluuebunny/crossref_metadata_2025 huggingface dataset:
Cofactor Activity in Placental Microvilli for the Cleavage of Complement Component 3 by Factor I
Authors: Vanderpuye Oluseyi A.
Published: April 2020

Human placental microvilli are in direct contact with maternal blood and the activity of complement components of the immune system must be regulated on this plasma membrane to prevent adverse effects to the fetus. A key point for regulation of the complement cascade is the proteolytic inactivation of complement component 3 which has become fixed on cell membranes by Factor I which requires a membrane bound cofactor. CD46 is the most widely distributed integral membrane protein cofactor for Factor I and is abundant in placental microvilli. An assay was developed for cofactor activity for Factor I that involved coupling of biotinylated methylamine‐ activated C3 (C3ma) to thiolpropyl agarose as a substrate. Microvilli from the surface of placental trophoblast cells are readily isolated by subcellular fractionation. The immobilized C3ma substrate was incubated with Triton X‐100 solubilized microvilli or other potential cofactor sources in the presence of Factor I. Cleavage of the C3ma was detected by SDS gel electrophoresis followed by electrophoretic transfer to nitrocellulose, blotting with alkaline phosphatase‐conjugated avidin and detection with an alkaline phosphatase substrate. It was found that cofactor activity was detectable in Triton X‐100 solubilized placental microvilli and platelets. However, a soluble form of CD46 that was not sedimented by centrifugation at 100,000g was associated with microvilli and also had co‐factor activity. In addition it was found that either CD46 or Factor I could first be bound to the immobilized C3ma and that cleavage could then be initiated upon addition of the missing component. The cofactor activity in placental microvilli could be removed by immunoabsorption with polyclonal or monoclonal antibodies to CD46 suggesting that this was the main source of cofactor activity for intrinsic C3 inactivation on placental microvilli.

In conclusion, CD46 was found to be the major if not only cofactor on placental microvilli for Factor I inactivation and existed in membrane bound and soluble forms associated with microvilli. Either of Factor I or its cofactor CD46 can bind to C3ma and the missing component can be added to form an active ternary complex.
Augmenting Medical Neuroanatomy Cadaveric Dissection with DiceCT Imaging
Authors: Bertolino Blake , Speer Weston , Gignac Paul , O’Brien Haley
Published: April 2020

Dissection of the brain and central nervous system for medical education is an informative but often wholly destructive process, leaving students with little reference material from which to review neural structures and their anatomical relationships. Here, we explore the use of diffusible iodine contrast enhancement followed by high‐resolution microCT (diceCT) to combine destructive physical dissection with more permanent digital dissection. DiceCT methods render soft tissues radiodense to X‐rays, emulating the tissue discrimination of magnetic resonance imaging (MRI). Such imaging studies provide students the opportunity to identify and interpret pathological findings in ex‐vivo neural tissues. To identify best‐practices for incorporating diceCT imaging into the medical neuroanatomy lab, we stained formalin‐preserved human brains with three concentrations of iodine (3, 4, and 5% aqueous I2KI) for 3 weeks, replacing the iodine solutions after the first and second weeks. All brains were microCT scanned at 63‐micron resolution, and the resulting image stack was rendered in Avizo version 2019.2. The stained brains were then bisected, and one hemisphere was de‐stained for 4 weeks by alternating baths of 1% sodium thiosulfate with deionized water each on a week‐long cycle. All concentrations of iodine were effectively de‐stained using this protocol. Students then performed comparative physical dissections on never‐stained, stained only, and stained‐then‐de‐stained brains, and comparative digital dissections were performed on brains of each iodine concentration. In physical dissection, stained only brains had few advantages—although the dark iodine stain enhanced some fiber tracts, specimens were smaller, physically difficult to manipulate, and harbored subcortical nuclei (e.g. the basal ganglia) that were more difficult to identify. Dissection of never‐stained and de‐stained brains was found to be equivocal, with de‐stained brains regaining much of their original size. In digital dissection, the 5% aqueous I2KI solution provided the clearest discrimination between structures, and pathologies such as age‐related atrophy and hydrocephalus were apparent. In addition to leaving a more permanent imaging study record of nervous tissue specimens, digital dissection allows for 3D printing of brain regions. These benefits were positively received by students, and we recommend incorporation of rapid, inexpensive tissue staining and tomography to enhance participation and clinical skills in the anatomy lab.

Support or Funding Information

Federal Work Study, OSU Center for Health Sciences
Claudin‐1 responsiveness through modulation of the inflammatory status in the Caco‐2 model epithelium.
Authors: Largoza Gabrielle E. , Carr Nicholas R. , King Jonathan M.
Published: April 2020

The use of hydrocortisone in association with indomethacin is a known risk factor for spontaneous intestinal perforation (SIP) in extremely low birthweight infants. While hypoxia, inflammation, and hydrocortisone have been found to independently alter both tight junction functionality and formation, the concomitant effects of inflammation, hydrocortisone, and indomethacin have yet to be well defined. Through the use of the Caco‐2‐BBe cell line as an in vitro model of the intestinal epithelial barrier, we tested the effects of the pro‐inflammatory agents TNF and IFNγ and the anti‐inflammatory agents indomethacin and hydrocortisone on barrier integrity and tight junction protein expression. Confocal imaging and cellular ATP analysis after 24 and 48 hours of exposure to the inflammatory agents both showed no significant changes in cellular proliferation or ATP synthesis, respectively. Western blot analysis found that claudin‐1 protein expression and localization is affected by inflammatory cytokines. Live cell confocal imaging of eGFP‐claudin‐1 Caco‐2 cells found that addition of pro‐inflammatory agents or the addition of anti‐inflammatory agents resulted in rapid dynamic junctional reorganization. When both the pro‐ and anti‐inflammatory agents were added, this pattern of reorganization was also seen. However, the concomitant effects of the combined pro‐ and anti‐inflammatory agents resulted in a profound inability to recapitulate its original claudin‐1 configuration. The lack of an effect on cellular proliferation and ATP synthesis paired with the alterations in claudin‐1 protein levels and localization suggests that claudin‐1 may be a potential target in better understanding the progression of spontaneous intestinal perforation.

Competing Interest Statement

All authors have indicated they have no potential conflicts of interest to disclose. The view(s) expressed herein are those of the author(s) and do not reflect the official policy or position of Brooke Army Medical Center, the U.S. Army Medical Department, the U.S. Army Office of the Surgeon General, the Department of the Air Force and Army, Department of Defense or the U.S. Government.

Support or Funding Information

Trinity University Biology Department
Tumor Growth in the Skin during Anagen and Telogen Phases of the Mouse Hair Cycle
Authors: Colombo Dan , Das Ridhima , Almazyad Asma , Gao Yao , Bielenberg Diane R.
Published: April 2020

Testing novel anti‐cancer drugs requires preclinical cancer trials, often performed in mice. The host tumor microenvironment (TME), defined as the cells surrounding or infiltrating the tumor such as stromal cells and immune cells as well as their secreted proteome, may affect tumorigenicity and progression. Most experimental tumor models are performed cutaneously. The skin is the largest organ in the body and undergoes morphological and cellular changes during each phase of the hair cycle: anagen (growth), catagen (regression), and telogen (resting). The rodent hair cycle differs from that of humans in that it is synchronized and predictable. We hypothesize that the TME and its associated protein milieu may differ in each phase of the mouse hair cycle. To examine the physical variations between anagen and telogen phases, C57Bl/6J mice were photographed before and after depiling (which induces a new hair cycle) or clipping (which does not induce a new hair cycle). Skin biopsies were obtained from these mice during anagen or telogen and analyzed histologically by hematoxylin and eosin staining and by immunohistochemistry to detect dividing cells (Ki67‐positive) and blood vessels (CD31‐positive). Epidermal and dermal thickness, proliferative index and vascular density were quantified and compared between the groups. Unbiased differential proteome profiles were compared between anagen and telogen skin samples using a mouse angiogenesis antibody array. Select proteins were also compared between the groups using immunoblotting and ELISA. Lastly, syngeneic B16F10 metastatic mouse melanoma cells were injected intradermally into the dorsal flank of (shaved) male or female mice during the telogen phase (8–11 weeks old) or the anagen phase (12–15 weeks old) of the hair cycle. Tumor incidence and growth rates were compared between the groups. Our data indicate that cells in the skin including fibroblasts, keratinocytes, and endothelial cells are highly proliferative during anagen but quiescent during telogen. However, the relative expression of angiogenesis‐related proteins is higher in the telogen phase compared to anagen phase. We conclude that the TME during anagen and telogen phases are distinct. The variable TME may account for heterogeneity between animal experiments or disparate preclinical results among laboratories ultimately leading to difficulties in replication and/or poor translation to human trials.
Extrahepatic Biliary Tract Anatomy: A Review
Authors: Giron Felipe Manuel , Ramírez-Buchheim Daniela Paola , Pizano Alejandro Manuel , Rueda-Esteban Roberto Javier
Published: April 2020

Laparoscopic cholecystectomy is one of the most common surgical procedures in general surgery. Significant gaps in knowledge still exist and even with clinical expertise, there might be limitations. Some of these are exploration restrictions during laparoscopic approach, visualization impairments and distortion of the usual arrangement of structures because of local inflammation, among others.

Given the wide variability in anatomical presentation and considering that even in 56% of the population there is a close relationship to main vascular structures, intraoperative injuries might occur leading to higher rates of complications, longer surgical time and increased morbidity.

Our aim is to describe the most common and relevant anatomic variations in the extrahepatic biliary tract (EBT). A systematic review of the literature was pursued using the MeSH terms: “Biliary Tract/abnormalities”, “Bile Ducts and Extrahepatic/abnormalities”. Only articles written in Spanish and English and limited to the last 20 years were considered.

The anatomical structure considered normal for the EBT is described in roughly 60% of the population. The most variable structures according to included manuscripts are the left and right biliary ducts and the most prevalent variations are: posterior branch of the right hepatic duct draining into the left hepatic duct, triple confluence of the intrahepatic ducts, and anterior or posterior branch of the right hepatic duct draining into the common hepatic or cystic duct.

As mentioned before the awareness and comprehension of EBT variations is crucial in avoiding complication, morbidity and achieving optimal outcomes in diagnostics and therapeutic procedures as reducing intraoperative iatrogenic injuries and considering the need of noninvasive techniques for EBT identification for a better surgical planning.
Expanding an anatomy division during a curricular reduction in medical anatomy ‐ Turn that frown upside down!
Authors: Lambert H. Wayne , Ressetar Holly G.
Published: April 2020

At West Virginia University (WVU) School of Medicine, a new curricular revision has reduced the Human Structure course, which includes gross anatomy, ultrasound, histology, and embryology, from a 14 week course to a 9 week course for first year medical students. This curricular modification also limits student contact to 15 hours a week, moves the Human Structure course from the spring to the fall semester, reschedules exam days from Mondays to Fridays, limits the coverage of new course content during exam weeks, and reduces the number of exams and assessments testing student learning. In response to these drastic changes, this new Human Structure course will reduce the number of lectures and laboratory (anatomy, ultrasound, and histology) sessions, change small group dissections to prosections, rotate students through the dissection laboratory in two groups to reduce contact time, and move a majority of the histology content to an online format, limiting the use of microscopes within the course. Throughout this transition period, the WVU Anatomy Division within the Pathology, Anatomy, and Laboratory Medicine Department has still been able to grow and remain relevant. Improvements within the Anatomy Division include, but are not limited to, recruitment of new faculty members, building two new dissection laboratories, securing funding for the construction of a new morgue to house the anatomical donors in the Human Gift Registry, and increasing the number of anatomy courses taught by faculty members within our Anatomy Division. Despite the reduction in coverage of the anatomical sciences to medical students, the demand for new anatomy courses has risen with the added benefit of teaching many new cohorts of students, including physician assistants, occupational therapists, and undergraduate students in exercise physiology, music therapy, and athletic training. More importantly, all anatomy courses taught within the WVU Health Sciences Center (HSC) will be moved under the direction of the Anatomy Division. Despite some bumps in the curricular landscape, anatomy instruction at the WVU HSC will continue to grow, offer new courses to a more diverse student population, and impact student learning.
Sex‐dependent Liver Volume Recovery and Hemodynamics Measured by Non‐Invasive Ultrasound Imaging after Partial Hepatectomy in Rats
Authors: Barnhart Benjamin , Kan Toshiki , Wessner Corinne E. , Eisenbrey John R. , Hoek Jan B. , Vadigepalli Rajanikanth
Published: April 2020

Initiation of liver regrowth after partial hepatectomy (PHx) is often linked to increased flow per unit tissue through the hepatic portal vein. Previous studies of liver hemodynamics have used surgical implants, however ultrasound‐based methods can non‐invasively measure blood flow, organ volume, and other physiological parameters.

Male and female Sprague‐Dawley rats were pair‐fed liquid diet containing ethanol or carbohydrate per Lieber DeCarli protocol for 5–6 weeks prior to a 70% PHx in which left lateral and medial lobes were surgically removed. A VEVO 2100 (VisualSonics, Toronto, Canada) high‐frequency ultrasound system with a motorized‐step ultrasound transducer was utilized to calculate liver volume using a 3D mapping software. Shear‐wave elastography measured liver stiffness with an Aplio i800 ultrasound system (Canon Medical Systems, Tustin, CA, USA). Color and Spectral Doppler evaluated blood flow velocity in the portal vein (PV) and the main hepatic artery (HA) at their entry into the right lobe. Laser coupled photoacoustic imaging aided in quantification of oxygen saturation in the liver tissue.

Liver regrowth as measured by liver volume was impaired in ethanol‐fed male rats as early as 48 hours after PHx, only recovering ~70% of initial volume by one week. In contrast, all other animals, including ethanol‐fed females, had recovered to ~100% of liver volume by one week post‐PHx. All animals showed increased PV and HA blood flow per liver volume immediately after PHx. Relative PV flow continued to increase by the 12‐hour time point, while relative HA flow had variable changes. Ethanol‐fed males had decreased ratio of PV/HA flow compared to all other animals prior to PHx. In the first 24 hours after PHx, relative HA flow decreased or remained stable for most groups, but increased for ethanol‐fed females. Liver stiffness increased immediately after PHx and again by 10 hours after PHx for all groups, though never reaching values that would suggest fibrosis. Stiffness remained elevated for at least 96 hours for all groups, and resolved by one week post‐PHx. Photoacoustic imaging showed liver tissue oxygen saturation fluctuating between 50–60% for all animals, with no discernable patterns.

Ethanol‐fed male rats had an impaired liver volume recovery response after PHx, however this ethanol‐mediated impairment was not seen in females. Interestingly, ethanol‐fed males were the only group to have a decreased PV/HA flow ratio prior to PHx, while ethanol‐fed females were unique in their prolonged increase in HA flow at 10 hours post‐PHx. Taken together, these results implicate increased HA flow as a compensatory mechanism in chronic ethanol‐fed rats.

Support or Funding Information

Supported by NIH NIAAA R01 AA018873, T32 AA007463 and NIBIB U01 EB023224.
Drug Discovery for Multiple Hereditary Exostoses
Authors: Weiss Ryan , Rother Sandra , Griglio Alessia , Oukoloff Killian , Heynen-Genel Susanne , Ballatore Carlo , Esko Jeffrey
Published: April 2020

Multiple hereditary exostoses (MHE) is an autosomal dominant disorder that results from heterozygous loss‐of‐function genetic mutations in exostosin 1 (EXT1) or exostosin 2 (EXT2). One in 50,000 children worldwide are affected by this relatively rare pediatric disease, which is characterized by osteochondromas that form next to the growth plates of skeletal elements. EXT1 and EXT2 encode the copolymerase responsible for biosynthesis of the backbone of heparan sulfate (HS), a cell surface polysaccharide. Heterozygous mutations in EXT1 or EXT2 lead to shortened HS chains in plasma and various tissues. Currently, there are no approved therapies for this disease; therefore, there is much interest in preventing and/or slowing the growth of exostoses through the development of small molecules that enhance HS levels on the cell surface. In this study, a high‐throughput phenotypic screen was developed to discover potential small molecule drug candidates that augment HS expression in mammalian cells. Screening of a compound library of ~57,000 molecules resulted in the discovery of several N‐aryl‐2‐aminothiazoles that displayed favorable “drug‐like” physicochemical properties, exhibited minimal cytotoxicity, and enhanced HS expression up to 3‐fold on the cell surface. Preliminary studies of structure‐activity relationships identified active analogs of the primary hit and key physicochemical properties required for their biological activity. Ongoing studies aim to expand the SAR studies, identification of a preferred candidate for in vivo studies, and finding the biological target for these compounds. Overall, these studies provide a promising new avenue for the development of novel therapies for MHE and other disorders involving glycosaminoglycan deficiencies.

Support or Funding Information

R01AR064202‐03 (to J.E.), K12HL141956 (to R.W.)
Establishment of screening for agents for improving dysgeusia using medaka
Authors: Tsutsumi Rie , Beppu Kana , Morio Atsushi , Sakaue Hiroshi , Tsutsumi Yasuo M.
Published: April 2020

Objects

Dysgeusia are caused by various factors such as chemotherapy, drugs, infection, and aging, and many people suffer. However, effective treatments for dysgeusia have not been established for drugs and foods. We have previously reported that the expression of lingual taste receptor T1R3 is reduced during chemotherapy induced dysgeusia. On the other hand, medaka, one kind of small fish, has many taste buds from mouth to gill, and the number is 5–6 times that of mice. In this study, we created a medaka that expressed the T1R3 gene with a fluorescent tag, and tried to visualize dysgeusia.

Methods

We established T1R3 knock‐in medaka using Crisper/cas9 system. The construct was microinjected into eggs immediately after medaka laying, and knock‐in was confirmed with a fluorescence microscope. It was confirmed that expression of T1R3 disappeared when reared in a solution containing an anticancer drug (5FU or platinum agent) using medaka of the F1 generation or later.

Results

We succeeded in producing a medaka that visualized the taste receptors by microinjecting fluorescently labeled T1R3 into medaka eggs. The expression of T1R3 disappeared when reared in a solution containing an anticancer drug whereas several amino acids such as glutamate and overeating were effective in maintaining and increasing T1R3 expression.

Conclusions

We established screening for agents for improving dysgeusia using medaka.

Support or Funding Information

This study was supported by Japan Society of Promotion Science #18K11075 to RT.
Molecular Interaction of Neuropilin‐1 and 3‐O‐sulfated Heparan Sulfate Modulates Angiogenesis‐dependent Tumor Growth
Authors: Painter Chelsea D. , Clausen Thomas M. , Park Jeeyoung , Vasquez Gabriel J. , Liu Jian , Gordts Philip L.S.M. , Esko Jeffrey D.
Published: April 2020

Neuropilin‐1 (Nrp1) acts as a coreceptor with Vascular Endothelial Growth Factor Receptor (VEGFR) to facilitate binding of its ligand, Vascular Endothelial Growth Factor (VEGF). Heparan sulfate (HS) has been shown to be a cofactor facilitating the interaction of Nrp1 with VEGFR/VEGFs, but the functional significance of this interaction has not been established. Previous studies have shown that Nrp1 binding to heparan sulfate is enhanced by 3‐O‐sulfation, a rare modification in which a sulfate group is installed at the C3 position of a glucosamine residue. Nrp1 mediates angiogenesis including branching and organization of vessels through its role in tip cell function and interactions with VEGFR/VEGFs. We hypothesize that heparan sulfate, particularly 3‐O‐sulfation, is critical for Nrp1 function in developmental and pathological angiogenesis. We have created a series of Nrp1 mutants to identify the specific amino acids that interact with heparan sulfate, particularly those that interact with the 3‐O‐sulfate group. Specific mutations in the b1b2 domains of Nrp1 decrease binding to heparin and cell surface heparan sulfate. These mutants also prevent heparin‐induced stabilization of the protein during thermal denaturation. Knock‐in mice expressing Neuropilin‐1 deficient in heparan sulfate binding (Nrp1HSBD) were created using CRISPR/Cas9 gene targeting. These mice are currently being evaluated for overall gross phenotype as well as proper branching and organization of developing vessels. Analysis of subcutaneous tumor formation revealed that Nrp1HSBD mice exhibit a reduction in tumor growth compared to wildtype controls. This finding suggests that the interaction of Neuropilin‐1 with heparan sulfate modulates its function in pathological angiogenesis.
Factors contributing to the equal forelimb and hindlimb stride lengths during quadrupedal walking
Authors: Johnson Laura E. , Zeininger Angel , Schmitt Daniel
Published: April 2020

The forward motion of terrestrial vertebrates can be divided into stride lengths (distance traveled by the body from touchdown to the next touchdown of a limb) which is the sum of the lengths of stance and swing phases for each limb. Those lengths are influenced by the anatomy of the limbs; longer limbs, for example, produce longer steps and strides. During typical quadrupedal walking, at least one forelimb and hindlimb are in contact with the ground at any given time. This means the fore‐ and hindlimbs (regardless of limb proportions) should have the same stride length, though the length of the phases (stance, swing) may differ, as long as they sum to equal stride lengths. In addition to the anatomical limb length (ALL), joint excursion patterns, which often differs between the forelimb and hindlimb can influence stance and step. Animals can behaviorally adjust their limb postures to change their effective limb lengths (ELL, the minimum distance between the shoulder/hip and wrist/ankle) in order to achieve the necessary stride length. Thus, we hypothesize that forelimb and hindlimb stride lengths should be equal, but this may be achieved in different ways and therefore all other parameters may vary possibly in relation to limb proportions.

To study this, we examined 69 quadrupedal walking bouts of four New World monkeys with differing ALL. Preliminary analyses indicate a close correlation of forelimb and hindlimb stride length (0.93, p<0.01), regardless of differences in ALL proportions. Factors that contribute to an equal stride length are more variable. Forelimb and hindlimb stance phase length were highly correlated (0.700, p<0.01), and forelimb and hindlimb swing phase length were highly correlated (0.834, p<0.01). Although significant, a low correlation of the length of swing and stance phase was found for both the forelimb (0.412, p<0.01) and the hindlimb (0.430, p<0.01). When modeling step length in stance by angular excursion and ELL, the model accounts for an average of 85% of the distance traveled. Taken as a whole, these data, especially the intralimb and interlimb patterns, suggest that the step and swing lengths are not easily predicted by limb anatomy or effective limb lengths alone. Thus, other factors must also contribute to step length and thus equal stride lengths, such as bending of the back and contributions made by hand and foot posture. Overall, these data indicate that although stride lengths do need to be equal between the forelimb and hindlimb, contributing parameters vary between strides and may vary by species anatomy and ecology.

Support or Funding Information

NSF BCS‐9222526
Re‐designing Biochemistry Laboratory to Implement BASIL CURE: from Structure to Function, from Student to Researcher
Authors: Goodman Anya L. , Jones Eric M. , Laubscher Andrea M. , Henry Jack , Craig Paul A.
Published: April 2020

Course‐based undergraduate research experiences (CUREs) have recently emerged as an alternative to traditional science laboratory instruction. Cross‐institutional faculty communities help crowd‐source curricular design and assessment, as well as facilitate data sharing and trouble‐shooting. As participants in the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) community, we re‐designed our upper level biochemistry laboratory course to incorporate CURE. Scientific objectives of BASIL focus on characterizing biochemical activities of proteins with known structure and unknown function. BASIL curriculum (freely available on GitHub
https://basilbiochem.github.io/basil/) includes modules on computational predictions of protein function, protein purification, and analysis of biochemical activity and kinetics.

Over the course of four years, our implementation has evolved to accommodate local needs and constraints and to address technical, logistical and pedagogical challenges we encountered along the way. We discuss this evolution in light of recommendations for CURE course design (Kloser et al., 2011) and the five dimensions of CURE (scientific practices, discovery, relevance, collaboration, and iteration; Auchincloss et al., 2014). Three main challenges for our implementation included 1) scaling of research training from the “apprentice” model to the classroom, 2) alignment of course learning objectives, activities and assessments with research goals to create optimal conditions for productive struggle, 3) student buy‐in.

CURE implementation presents challenges with timing of research experiments in a scheduled course, availability of equipment, management of reagent requests and hazardous waste. Instructional support staff (AML) was critical for overcoming a number of logistical, technical and pedagogical challenges. In addition, undergraduate students pursuing research projects outside of class contributed to the CURE by developing new protocols, testing new proteins or generating computational predictions using tools that were outside the scope of the course. To understand if/how students in our course develop laboratory research skills, we began assessing anticipated learning outcomes previously defined by Irby et al. (2018), focusing on students’ evaluation of protein purification experiments. Initial assessment results revealed learning difficulties and led to modifications in instructional activities, including use of simulated experiments. To gauge student frustration and to help students improve their metacognitive skills, we asked students to reflect on their experiences in short weekly structured journal entries. Students’ negative perceptions of failed experiments, potentially, rooted in fixed mindset, require additional intervention.

Support or Funding Information

Supported by NSF‐IUSE #1503676 to ALG, #1710538 to ALG and EMJ; 1503811 and 1709170 PAC.
To bee inside or outside? Impacts of overwintering environment on gene expression of the alfalfa leafcutting bee, Megachile rotundata
Authors: Cambron Liz D. , Yocum George D. , Greenlee Kendra J.
Published: April 2020

Diapause is a non‐feeding stage that many insects go through to survive the winter months. How nutrition is regulated during these months of overwintering is still unclear. Diapausing insects are exposed to environmental stressors including temperature stress, but responding can be energetically costly. With fixed resources, overall metabolism and insulin signaling are maintained at low levels, but it is unclear if those pathways change in response to seasonal temperature fluctuations. Our hypothesis is that insulin signaling is responsible for allocating energy in response to fluctuations in temperature during overwintering months. To test this hypothesis, we overwintered alfalfa leafcutting bees, Megachile rotundata, in either a lab setting at a constant 4°C or in the field in naturally fluctuating temperatures, collected samples monthly throughout the winter, and then measured expression of genes in the insulin pathway using nCounter analysis (NanoString Technologies Inc.). nCounter is a highly multiplexed single molecule counting technique. A Dunn’s test was performed to analyze the effects of time and location for all possible comparisons. Our results showed that several genes were differentially expressed over time and between locations. However, more changes were seen in field samples, possibly in response to environmental cues. With a hierarchical cluster, field samples showed three distinct month clades, possibly correlating with different developmental stages: diapause, post‐diapause quiescence, and direct development. Although several genes were differentially expressed, many of them were downstream of the insulin pathway (Foxo, MAPK, GSK3β). This could mean that components of the insulin pathway are being utilized to respond to temperature fluctuations in the field. Along with evidence from other studies, our results suggest that insects may use different mechanisms for diapause regulation in response to their environment. This plasticity in regulation may be achieved by using targets of other pathways such as that of the insulin pathway. There is no denying that pollinators are crucial for the health of our ecosystem, but before we can save the bees, we need a better understanding of how their overall physiology is impacted in a drastically changing environment.

Support or Funding Information

This research was funded, in part, by grants from the National Science Foundation Graduate Research Fellowship and an NSF Graduate Research Internship with the USDA‐ARS Fargo, ND, and NSF EPSCoR‐1826834 to KJG.
XRT: A Reproducible and Scalable Training System that Produces Students Capable of Conducting Novel Molecular Biology Research
Authors: Groshong Tyler L. , Fong Megan J. , Madejski Irene R. , Leeman Jacob T. , Sundaresan Avirath , De Luke
Published: April 2020

Many high‐school science education programs do not train students to be independent researchers that make novel contributions to the scientific community, which is arguably the primary goal of a scientist. There are many possible explanations for this paradox. Due to high student/teacher ratios, teachers are unable to give the individualized attention necessary to cultivate a scientist from a student. Class schedules, student motivation, and diverse intellectual support are also huge barriers. As a result, most high‐school programs struggle to establish a system that allows for university and graduate‐level laboratory education of a significant number of students.

Here, we report a curriculum that trains students to design, conduct, manage, lead, and present novel research projects, but also a program that is self‐sustaining and scalable. The system produces a three‐tiered system of research students, each with an increasing set of laboratory and troubleshooting, goal‐setting, and project management skills. This management system is scaffolded. It can be completed at different speeds. These teams design projects that are novel and real. Trained teams come together and systematically create projects that can not only sustain themselves and pursue novel intellectual discovery, but also projects that are able to expand and adapt through multiple years and to a changing number of students, independent of staffing.

Support or Funding Information

This work was supported by The Nueva School.
The importance of creating your own Anatomical Study Aids, from an educator’s perspective
Authors: Leighton Maria Ximena
Published: April 2020

For many years in the traditional medical education we have used books and atlases to teach anatomy. The students were presented with a lecture for that day’s Lab dissection and then went directly to the Anatomical Laboratory and follow the instructions in a dissector.

The atlases contain diagrams of everything they need to know but at the same time they have an important limitation showing the depth of the structures; which is very important for the students to be able to understand the three dimensional aspect of the human body.

Then, we added Atlases with photographs from dissected bodies which came out more than two decades ago. These ones have been very helpful teaching aids. However they also presented some challenges such as the difficulty by the students to follow the labels in many pictures and the bodies presented differed a lot from the bodies they were working on their dissections. As a results many students use them as a review tool.

Also now in the modern medical education we have incorporated electronic teaching aids and the students have easy access to internet, therefore they can google anything. Unfortunately not everything they can find is accurate.

Because of all these concerns, I started creating multimedia teaching materials tailored to the students at my institution. Using a cadaver embalmed in the same way as the one used by the students, I created dissecting videos using our own dissector. I also produced photographs which I am using now to create online reviews and many other materials for the students.

Creating my own material has helped me to show the students the structures in a more efficient way and most importantly has helped the students to have a better three dimensional understanding of the human body.

The opinions or assertions contained herein are the private ones of the author/speaker and are not to be construed as official or reflecting the views of the Department of Defense, The Uniformed Services University of the Health Sciences or any other agency of the U.S. Government.

Support or Funding Information

USUHS
Role of PCSK1N and GPR83 in salt resistance
Authors: Zheng Xiaoxu , Kumar Megha , Konkalmatt Prasad , Asico Laureano , Jose Pedro A , Armando Ines
Published: April 2020

Previous studies from our laboratory show that in mice increased expression/function of the orphan receptor GPR83 is associated with salt resistance. GPR83 is expressed in proximal and distal convoluted tubules of the mouse kidney. Global deletion or renal‐specific silencing of the GPR83 gene increases systolic blood pressure (SBP) and increased G protein‐coupled receptor kinase type 4 (GRK4) expression in the kidney. Recent evidence indicated that peptides derived from the Proprotein Convertase Subtilisin/Kexin Type 1 Inhibitor (PCSK1N) may be the endogenous ligands of GPR83. We hypothesized that these peptides are implicated in the regulation of blood pressure. Renal specific siRNA‐mediated silencing of PCSK1N in mice resulted in an increase in SBP (130±4 vs control wild‐type 100±1 mmHg, n=3/group; P<0.05); this was associated with decreased urinary Na+ excretion (P<0.05) and decreased renal cortex expression of GPR83, but increased NHE3 and Na+/K+‐ATPase expressions (P<0.05 all). The peptides PEN (proline, glutamic acid, and asparagine) and SAAS (serine, alanine, alanine, and serine) are derived from enzymatic breakdown of Pro‐SAAS. Renal restricted infusion of PEN (1 μg/day; 7 days) did not affect SBP, slightly decreased Na+ excretion and increased GPR83, GRK4, and NHE3 expressions (P<0.05 all) but did not affect Na+/K+‐ATPase expression. By contrast, renal‐restricted infusion of SAAS (1 μg/day; 7 days) in mice decreased SBP (82± vs 100± mmHg; P<0.05), Na+ excretion did not change but decreased the renal cortex expression of GRK4, GPR83, NHE3 and Na+/K+‐ATPAse (P<0.05 all). Furthermore, PCSK1N expression was increased by a high salt diet (4%NaCl) in non‐transgenic mice but was decreased in the salt‐sensitive GRK4 486V mice on the same diet. Our result suggested that Pro‐SAAS peptides may be involved in the development of salt sensitivity through gene‐gene interactions among PCSK1N, GPR83, and GRK4.

Support or Funding Information

This work was supported by NIH grant t R01DK039308 (P.A.J.);NIH grant R37HL023081(P.A.J.); NIH grant P01HL074940 (P.A.J.); NIH grant P01HL068686 (P.A.J.); NIH grant R01HL092196 (P.A.J., I.A.); NIH grant R01DK09098 (I.A.)
‘MUSCADINE GRAPE SKIN EXTRACT INHIBITS ANDROGEN‐INDEPENDENT PROSTATE CANCER CELL GROWTH, INDUCING CELL‐CYCLE ARREST, AND DECREASING MIGRATION BY TARGETING HEAT SHOCK PROTEIN 40
Authors: Brown Collis
Published: April 2020

Previously we demonstrated that muscadine grape skin extract (MSKE), a natural product, significantly inhibited androgen‐responsive prostate cancer cell growth by inducing apoptosis through the targeting of survival pathways. However, the therapeutic effect of MSKE on more aggressive androgen‐independent prostate cancer remains unknown. This study examined the effects of MSKE treatment in metastatic prostate cancer using complementary PC‐3 cells and xenograft model. MSKE significantly inhibited PC‐3 human prostate cancer cell tumor growth in vitro and in vivo. The growth‐inhibitory effect of MSKE appeared to be through the induction of cell‐cycle arrest. This induction was accompanied by a reduction in the protein expression of Hsp40 and cell‐cycle regulation proteins, cyclin D1 and NF‐kBp65. In addition, MSKE induced p21 expression independent of wild‐type p53 induced protein expression. Moreover, we demonstrate that MSKE significantly inhibited cell migration in PC‐3 prostate cancer cells. Overall, these results demonstrate that MSKE inhibits prostate tumor growth and migration, and induces cell‐cycle arrest by targeting Hsp40 and proteins involved in cell‐cycle regulation and proliferation. This suggests that MSKE may also be explored either as a neo‐adjuvant or therapeutic for castration resistant prostate cancer.
Does hypoxic response enhance overload‐induced muscle hypertrophy ?
Authors: Takada Hiroaki , Osana Shion , Kubota Takahiro , Nunomiya Aki , Nagatomi Ryoichi
Published: April 2020

INTRODUCTION

We recently established a conditional knockout mouse which exhibits hypoxic response under normoxia. Prolyl hydroxylase domain 2 (PHD2), an enzyme responsible for oxygen dependent hydroxylation of hypoxia inducible factor (HIF), turns HIF susceptible to ubiquitination and subsequent degradation by the proteasome. We previously examined the effect on endurance capacity of PHD2 conditional knockout mice (cKO). A 4‐week training effect on endurance time was more than 1.6 times enhanced in the PHD2 knockout mice (A Nunomiya, et al., Acta Physiol, 2016). Blood flow restriction during strength training are known to facilitate muscle hypertrophy. How hypoxic response is involved in the process of muscle hypertrophy is yet to be clarified. Thus, we hypothesized that overload induced muscle hypertrophy may be enhanced in the PHD2 knockout mice.

METHODS

We used tamoxifen driven PHD2 cKO mice. After intraperitoneal administration of tamoxifen for 1 week, the cooperative muscles (gastrocnemius and soleus) for the plantaris muscle of the right leg were excised for overloading. Seven days later, we examined the weight, size, fiber‐type composition of the plantar muscles as well as the protein synthesis pathway such as the activation of the mTOR pathway and blood composition such as red blood cell count, VEGF (Vascular endothelial growth factor, EPO (Erythropoietin)).

RESULTS

Our results show that overloading induced plantaris muscle hypertrophy as compare to the control leg plantaris muscle. The hypertrophy was remarkably enhanced in the PHD2cKO(32% in control vs 83% in PHD2 cKO) .

CONCLUSIONS

Hypoxic response mediated by HIF pathway enhanced overload‐induced muscle hypertrophy.
“Meet the Patient” Sessions: An Effective Pedagogical Approach for Teaching Pharmacology to Pre‐clinical Medical Students
Authors: Athauda Gagani , De La Camara Frances , Shopa Keith , Krieger Diane , Toonkel Rebecca Lyn , Moulik Sabyasachi
Published: April 2020

Introduction

Opportunities for patient involvement in teaching pharmacology to pre‐clinical medical students is limited. At our institution, two “Meet the Patient” sessions were incorporated into the Period 2 Endocrine System course as a novel pedagogical tool. This method of teaching invites students to approach content in a clinically oriented fashion while meeting and discussing their learning with a live patient. In this study, we investigated student satisfaction and the effect of these “Meet the Patient” sessions on medical student pharmacology knowledge acquisition as assessed by MCQ final exam performance.

Methods

All second‐year students (Class of 2021, n=117) in the Endocrine Systems course participate in two mandatory “Meet the Patient” sessions focusing on Diabetes Mellitus and Pituitary/Adrenal Disorders. Prior to the sessions, students are required to view short pre‐recorded modules (covering pathophysiology and pharmacologic management) and to take a short readiness quiz. Sessions occur in the lecture hall, with students working in pre‐assigned groups of three. During the session, the facilitator briefly introduces the patient and allows students to ask probing questions to flesh out the history. Students then work together to complete guided in‐class learning questions. Student satisfaction was assessed anonymously through end of course evaluations (5‐point Likert scale). Pharmacology knowledge acquisition was assessed by student performance on session‐related pharmacology MCQs included on the internally developed final course exam and compared with student performance on all non‐session‐related MCQs on the same exam. Unpaired two‐tailed T‐tests (unequal variance) were used to compare the means.

Results

Mean agreement (agreed or strongly agreed, n=105) with statements that the sessions: “improved my understanding of management,” “improved my understanding of pathophysiology,” “helped me better understand the diagnosis,” “helped me understand the implications of the disease,” and that “I am more likely to retain information presented in this format than through traditional lecture” was found to be 91.43%, 87.63%, 90.48%, 93.34%, and 88.57%, respectively. Mean performance on session‐related pharmacology MCQs included on the internally developed final course exam (n=5 questions) was found to be 97% (SD 3.67) as compared with 85% (SD 17.97) for mean performance on all non‐session‐related MCQs (n=25 questions) on the same exam (p=0.005).

Conclusion

“Meet the Patient” style sessions are an effective tool for teaching pharmacology to pre‐clinical medical students. These sessions are well received by students and may lead to improved knowledge acquisition. Further study is needed to assess effects on retention and to explore potential explanations.
E‐Cigarette Use Increases Susceptibility to Bacterial Infection by Impairment of Human Neutrophil Chemotaxis, Phagocytosis and NET Formation
Authors: Corriden Ross , Moshensky Alexander , Bojanowski Christine , Meier Angela , Chien Jason , Nelson Ryan K. , Crotty Alexander Laura E.
Published: April 2020

Although E‐cigarettes are frequently portrayed as a safer alternative to conventional cigarettes, a growing body of evidence suggests that the use of these devices may adversely affect host defense against infection. Neutrophils, critical effector cells of the innate immune system and early responders to sites of infection, may represent a particularly important cellular target of E‐cigarette vapor components. Using primary human neutrophils isolated from healthy donors, we evaluated the impact of electronic vapor extract (EVE) exposure on multiple aspects of neutrophil cellular physiology, including chemotaxis, reactive oxygen species (ROS) production, generation of Neutrophil Extracellular Traps (NETs) and phagocytosis. Using a transwell chamber approach, we found that exposure to E‐cigarette vapor‐infused media (EVE) reduced neutrophil chemotaxis in response to the chemoattractant f‐Met‐Leu‐Phe (fMLP) by up to 4.2 fold (p < 0.001). Staining with the fluorescent actin probe rhodamine phalloidin revealed aberrant actin cytoskeleton polarization in EVE‐treated, fMLP‐stimulated neutrophils. Neutrophil membrane fluidity was also altered by EVE exposure, with holotomographic microscopy revealing alterations in cellular morphology in EVE‐treated neutrophils. ROS production was reduced in EVE‐treated neutrophils, and ROS‐dependent production of bactericidal extracellular traps was reduced by up to 3.5 fold (p < 0.01). Interestingly, this reduction in NET production (NETosis) was observed in neutrophils treated with EVE produced using propylene glycol‐based E‐cigarette liquid formulations both with and without nicotine. The rate of neutrophil phagocytosis, quantified using fluorescent E. coli/S. aureus bioparticles, was also reduced by up to 47% in EVE‐treated neutrophils (p < 0.05). To determine how such alterations of immune cell function might affect host response to infection, a physiologic mouse model of chronic E‐cigarette exposure and sepsis was utilized. In this model, E‐cigarette vapor inhalation led to reduced neutrophil migration into infected spaces and a higher burden of Pseudomonas aeruginosa. Taken together, these findings provide evidence that E‐cigarette use adversely impacts the innate immune system, potentially placing E‐cigarette users at higher risk for dysregulated inflammatory responses and invasive bacterial infections.