bluuebunny/crossref_metadata_2025 huggingface dataset:
Losartan prevents the mesangial cell‐myofibroblast transdifferentiation induced by high glucose and angiotensin II
Authors: Arnoni Carine Prisco , Maquigussa Edgar , Pereira Luciana Guilhermino , Boim Mirian Aparecida
Published: June 2021

This study evaluated the effect of losartan on the regression of the mesangial cell to myofibroblast transdifferentiation (MMT) induced by high glucose (HG) environment and by AngII. Mesangial cells (CM) were stimulated with HG (30mM) and AngII (10‐7M) or HG and AngII during 4 (4dG+AngII) or 6 (6dG+AngII) days to induce the myofibroblast transdifferentiation. To evaluate the regression process, the group 6dG+AngII was treated in the last 48 hours with losartan (10‐4, 10‐5 e 10‐6M). The expression of the components of the mesangial matrix including collagen I and IV, and fibronectin, the growth factors such as TGF‐â and connective tissue growth factor (CTGF) and the expression of smooth muscle á‐actin (SMAá), a marker of myofibroblast, was evaluated by PCR and Western blot. Matrix components and growth factor synthesis were increased in group 4dG+AII but the SMAá expression was significant in group 6dG+AII, when the cells also presented a high migration capacity, characterizing the MMT. Losartan (10‐4M) reversed the increase in SMAá expression and the cellular migration indicating a preventive effect on the MMT. These results suggest that the AngII inhibition can be a potential strategy, not only to avoid the progression of the glomerulosclerosis, but also to reverse the fibrogenic process.
Rapid Lysosome Fusion to Lipid Raft Clusters and Endothelial Dysfunction Mediated by VAMP‐2 in Coronary Arterial Endothelial Cells
Authors: Han Weiqing , Xia Min , Zhang Fan , Li Pinlan
Published: June 2021

Lysosome fusion mediates acid sphingomyelinase translocation to and activation in plasma membrane to form membrane lipid raft (LR) signaling in coronary arterial endothelial cells (CAECs). The molecular mechanism mediating this lysosome fusion to cell plasma, however, remains poorly understood. The present study attempted to evaluate whether vesicles associated membrane proteins‐2 (VAMP‐2) mediates lysosome fusion and LR clustering, leading to endothelial dysfunction in CAECs. By immunohistochemistry, VAMP‐2 was found to be abundantly expressed in the endothelium of bovine coronary arteries. Fluorescent confocal microscopy showed that VAMP‐2 was significantly aggregated with lysosome marker lamp‐1 and LRs when the CAECs were stimulated by FasL, a well‐known LR clustering stimulator. Using FM1‐43 quenching and dequenching technique, lysosome fusion to plasma membrane in response to FasL was blocked by VAMP‐2 inhibitor, tetanus toxin. In addition, fluorescence resonance energy transfer (FRET) detection demonstrated that Fas L‐induced FRET between LRs marker and VAMP‐2 increased by 21.2 ± 3.3 during FasL stimulation. Functionally, inhibition of VAMP‐2 significantly restored FasL‐induced endothelial dysfunction in coronary arterial rings. In conclusion, VAMP‐2 on lysosome membrane in CAECs is critical to the lysosome fusion to LR areas of plasma membrane in CAECs and this VAMP‐2‐mediated lysosome fusion may contributes to FasL‐induced endothelial dysfunction (Supported by NIH grants HL057244, HL091464 and HL075316).
Soluble endoglin inhibits breast cancer cell proliferation
Authors: Gingery Anne , Nelson Adam M , Soldner Emma L B , Gilbert Jeffrey S
Published: June 2021

While recent studies indicate preeclampsia may be associated with reduced incidence of breast cancer, the exact mechanisms underlying this observation remain unknown. Soluble endoglin (sEng) is one of the factors increased due to placental ischemia in preeclampsia and may play a role in reduced incidence of breast cancer by altering TGFβ signaling. Previously we have shown that serum from our rat model of preeclampsia inhibits growth of breast cancer cells independent of estrogen receptor status. We hypothesized that sEng inhibits proliferation of breast cancer cells via MAPK and PI3K/Akt pathways. We examined the effects of sEng on MCF‐7 and LCC9 cells in vitro. MCF‐7 cells are estrogen sensitive, while LCC9 are non‐responsive to estrogen. sEng treatment (20 ng/mL) reduced proliferation of MCF‐7 (65.6% ‐ 24h) and LCC9 (68.2% ‐ 24h) breast cancer cells. Phospho‐Akt (47, 57, 76 %) and phosph‐Erk1/2 (28, 44, 57 %) activation was reduced in MCF‐7 cells at 5, 10 and 30 minutes post‐treatment with sEng. Total Akt and MEK protein levels remained constant, whereas total Erk protein levels decreased within 10 minutes of treatment with sEng. Taken together sEng may act by attenuating TGFβ signaling thereby inhibiting proliferation of MCF‐7 breast cancer cells through the Akt and Erk signaling pathways. The present data are a step towards identifying cellular mechanisms that may be exploited to develop novel therapeutic regimens.
A calcium‐induced calcium‐release mechanism supports Luteinizing Hormone (LH) induced testosterone secretion in mice Leydig cells
Authors: Varanda Wamberto Antonio , Franci Celso Rodrigues , Costa Roberta Ribeiro
Published: June 2021

Binding of LH to a G Protein Coupled Receptor in the plasma membrane, results in a primary increase in cAMP and in the intracellular calcium concentration ([Ca2+]i in Leydig cells. Immunocytochemical labeling with specific antibodies show the presence of three distinct isoforms of both ryanodine receptors (RYRs) and inositol 1,4,5‐trisphosphate receptors (IP3Rs). Measurements of intracellular calcium activity, using the fluorescent calcium sensitive dye Fluo3 and confocal microscopy, demonstrate that these receptors are involved in a calcium induced calcium release mechanism (CICR), responsible for the amplification of the initial Ca2+ influx through plasma membrane T‐type calcium channels (CaV3), induced by LH. The RYRs and IP3Rs are functional, as judged both from their activation by caffeine and IP3 and block by ryanodine and 2‐APB, respectively. Nevertheless, the intracellular global calcium changes resulting from treatment of the cells with LH are readily blocked 100 μM ryanodine but not by 2‐APB or xestospongin C. Besides this, blockage of the ryanodine receptor inhibits both the hormone‐induced [Ca2+]i transients and steroid secretion by Leydig cells. These results not only broaden the understanding of the role played by calcium in Leydig cells but also show, for the first time, that ryanodine receptors have a crucial role in determining secretion of testosterone by the testis.

Financial Support: FAPESP, CNPq
Phosphorylation of TonEBP/OREBP by cyclin dependent kinase 5 (CDK5) mediates its NaCl‐induced nuclear translocation
Authors: Gallazzini Morgan , Heussler Gary E , Williams Chester K , Burg Maurice B , Ferraris Joan D
Published: June 2021

High NaCl increases TonEBP/OREBP nuclear localization. We find that high NaCl increases phosphorylation of TonEBP/OREBP‐T135, located in its Auxiliary Export Domain (amino acids 132–156). Mutation of T135 to alanine decreases high NaCl‐induced nuclear localization of TonEBP/OREBP. T135 is in a consensus site for phosphorylation by protein kinase CDK5. High NaCl increases phosphorylation of CDK5‐Y15, indicating increased activity. Inhibitors of CDK5 and Kinase Dead (KD) dominant negative CDK5 reduce high NaCl‐induced nuclear localization of TonEBP/OREBP. Furthermore, CDK5 associates with TonEBP/OREBP (coimmunoprecipitation) and directly phosphorylates T135 in vitro. Over expression of CDK5 KD strongly decreases phosphorylation of T135. We do not find any regulation of TonEBP/OREBP transactivating activity by CDK5. We conclude that high NaCl stimulates CDK5 kinase activity which in turn contributes to nuclear localization of TonEBP by phosphorylating it on T135.

This research was supported by the Intramural Research Program of NHLBI.
High NaCl or urea inhibits glycerophosphocholine (GPC) phosphodiesterase (GDPD5) activity and reduces phosphorylation of GDPD5‐T587
Authors: Topanurak Supachai , Gallazzini Morgan , Ferraris Joan D , Burg Maurice B
Published: June 2021

Glycerophosphocholine (GPC) is an osmoprotective renal medullary organic osmolyte. We previously found that high NaCl or urea (hyperosmolality) increases GPC and that post translational inhibition of the phosphodiesterase (GDPD5) that degrades GPC contributes to this increase. The purpose of the present studies was to identify the post translational modification. We find that hyperosmolality reduces phosphorylation of GDPD5 (less retardation in Western blots of phospho‐affinity polyacrylamide gels). Mass spectrometry (LTQ‐iontrap with targeted ion selective label‐free quantification) demonstrates phosphorylation of GDPD5 on T587 which is decreased by hyperosmolality. Current experiments are testing the role of phosphorylation at that site on GDPD5 activity.
PP2R2C is involved in the resensitization of the dopamine D1 receptor in human renal proximal tubule cells.
Authors: Zhang Yanrong , Yang Yu , Villar Van Anthony , Pascua Annabelle , Armando Ines , Jose Pedro A
Published: June 2021

Dopamine is important in the regulation of blood pressure, sodium balance, and renal function acting through D1‐like and D2‐like dopamine receptors. Protein phosphatase 2A (PP2A) is a heterotrimeric serine/threonine phosphatase. There is tissue specific expression of the B subunits of PP2A; human renal proximal tubule cells express B55γ (PP2R2C, γ isoform, B regulatory subunit) and PP2A B56 γ (□ isoform, B′ regulatory subunit). We have reported that dopamine D1 receptor (D1R) dephosphorylation, recycling to the cell surface membrane, and resensitization are dependent on PP2A activity (Kidney Int. 2006;70:1072–9). We aimed to determine the role of PP2R2C in the spatial and temporal events involved in the resensitization of D1R in human renal proximal tubule cells. PP2A B (entire B regulatory subunit), PP2A C (catalytic subunit) and PP2R2C subunits were found to be expressed in the cytosol of immortalized human renal proximal tubular cells by confocal microscopy (n=3). Co‐immunoprecipitation studies demonstrated that the D1R interacts with PP2A B, PP2A C and PP2R2C subunits (n=3). Treatment with the D1‐like agonist fenoldopam (FEN, 1 □M) decreased expression of PP2A B (15%; n=3) and PP2A B56γ (30%; n=3) but increased that of PP2R2C (25%: n=3). Treatment with FEN for 15 min increased total phosphatase activity (18%; n=2). Upon FEN stimulation the D1R internalized and co‐localized with of PP2R2C (n=3). These results suggest that PP2A and PP2R2C are involved in the regulation of the resensitization and trafficking of the D1R.

This work was supported in part by grants from the National Institutes of Health, HL68686, HL23081, HL074940, HL092196 and DK39308
Regulation of mitochondrial function by caveolin‐3
Authors: Moreno Ana L. , Kidd Michael W. , Panneerselvam Mathivadhani , Roth David M. , Patel Hemal H.
Published: June 2021

Caveolae, cholesterol ‐rich invaginations of the plasma membrane, are scaffolding domains that contain caveolins, proteins involved in signaling processes in the heart. Cardiac pathologies are characterized by mitochondrial dysfunction. Mitochondria act as buffers to respond to cell stress and determine cell survival or death. Caveolins, specifically caveolin‐3 (Cav‐3), may modulate mitochondrial function during stressful events. We hypothesize that overexpression of Cav‐3 in mouse myocardium modulates mitochondrial function. Three‐month old hearts from wild‐type or Cav‐3 overexpressing (Cav‐3 OE) mice were excised, then mitochondria were isolated and purified. Pure mitochondria were subjected to a calcium challenge once per minute until the mitochondria no longer became polarized, a sign of mitochondrial dysfunction. Mitochondria from Cav‐3 OE mice were more tolerant to calcium loading suggesting that these mitochondria are more robust and may be more efficient in tolerating stress. Also, Cav‐3 OE cardiac myocytes had reduced superoxide generation in response to hypoxia/reperfusion injury. These results imply that Cav‐3 overexpression plays a role in preservation of mitochondrial function.
Caveolin and the aged myocardium
Authors: Kidd Michael W. , Reichelt Melissa E. , Peart Jason N. , Niesman Ingrid R. , Head Brian P. , Headrick John , Roth David M. , Patel Hemal H.
Published: June 2021

The aged heart is more susceptible to ischemic injury than the young heart. We hypothesized that loss of efficiency via reduced cellular scaffolds leads to this susceptibility. We examined the contribution of caveolin‐3 (Cav‐3), a lipid‐raft scaffolding protein, to ischemic tolerance in aged myocardium. Cav‐3 expression was reduced in aged hearts. Cardiac‐specific expression of Cav‐3 improved ischemic tolerance in both young (3 months) and aged (24 months) hearts. Aged WT hearts exhibited significant dysfunction, recovering only 20–25% of force and exhibiting almost 2‐fold higher diastolic pressure. Overexpression of Cav‐3 profoundly improved outcome in aged hearts with 60–65% recovery of pressure development and end diastolic pressure in the normal range. These data reveal a parallel decline in cardiac ischemic tolerance and Cav‐3 expression, and indicate that increasing Cav‐3 expression can eliminate age‐related ischemic intolerance. Cav‐3 may serve as a novel therapeutic target to limit dysfunction associated with cardiac aging.
Re‐expression of p27 inhibits proliferation of endometrium cells from patients with endometriosis by modulating cell cycle‐related protein expression
Authors: Camargo‐Kosugi Cíntia Meirelles , Secco Mariane , Vassallo Paula Frizera , D'Amora Paulo , Sato Helio , Schor Eduardo , Zatz Mayana , da Silva Ismael Dale Cotrim Guerreiro , Gonçalves Giovana Aparecida
Published: June 2021

Endometriosis is a benign gynaecologycal disease defined as the presence of functional endometrial implants out of uterus, mainly in pelvis. The cyclin‐dependent kinase inhibitor p27kip1 seems to play a critical role in this process. Having this in mind, in the present study we investigated whether the re‐expression of p27kip1 could modulate cell proliferative response in the endometrium of women with endometriosis. First generation bicistronic adenovirus: AdCMVhp27IRESEGFP (Adp27) and AdCMVNull (AdNull) were engineered. After transduction, the re‐expression of p27kip1 was followed by a significant decrease in proliferation of endometrium cells obtained from women with endometriosis to a very close level of controls. In an attempt to better understand this anti‐proliferative effect, we also evaluate the expression of cell cycle‐related proteins (p16, p21, p27 and p53) in these cells. Results showed that p16, p21, p27 and p53 protein levels were markedly increased following Adp27 transduction. These findings further confirm our hypothesis that the lack of p27 expression in the topic endometrium of women with endometriosis might be one of the reasons that support proliferation of this tissue in places other than uterus. In addition, our results bring the interesting possibility for the use of Adp27 as a future strategy for the treatment of endometriosis.
Effects of hindlimb unweighting on estrogen replete and deficient mice
Authors: Crissey Jacqueline Marie , Ferreira J Andries , Brown Marybeth
Published: June 2021

Skeletal muscle atrophy is often debilitating. In women the decline in lean muscle mass accelerates at the onset of menopause due in part to lower estrogen (E2) levels. We evaluated the role of E2 to regulate skeletal muscle mass and maintain contractile function over 14 days of hindlimb unloading (HU) and 3 days of reloading (REC) in E2 replete (estrogen receptor‐α knock‐out, ERαKO) and E2 deficient (aromatase knockout, ARKO) mice, compared to wild type (WT) animals. Mice were separated into 3 groups: 1) CON: ambulatory controls, 2) HU: 14days of HU, 3) REC: 14 days of HU + 3 days REC. We observed differential effects on body weight (BW) after HU and REC. BW significantly decreased in ARKO mice (−10.9%) and ERαKO (−10.5%) after HU, while BW of WT mice was not different. Neither ARKO nor ERαKO recovered to pre‐HU BW values following REC. Expected declines in muscle mass of soleus (SOL), plantaris (PLAN), gastrocnemius (GAS), and tibialis anterior (TA) in WT mice after HU (Table 1) were observed. ARKO mice trended towards losing more muscle mass during HU and recovered slower than WT.

Percent atrophy of muscle mass loss in HU & REC compared to CON.

WT
SOL
PLAN
GAS
TA

HU
−38
−20
−15
−11

REC
−18
−6
−6
−2

ARKO
SOL
PLAN
GAS
TA

HU
−43
−30
−23
−12

REC
−34
−17
−19
−15

ERαKO
SOL
PLAN
GAS
TA

HU
−46
−9
−15
−4

REC
−25
−7
−7
−8

The ERαKO muscle mass profile resembled that of WT mice, suggesting that the presence of E2 rather than the absence of ERα mediates estrogen effects during HU and REC. These data suggest that estrogen deficiency slows skeletal muscle mass recovery. Support: HD‐058834 & University of MO Life Sciences Fellowship.
Substrate‐dependent localization of transglutaminase 2
Authors: van den Akker Jeroen , Afink Gijs B , van Weert Angela , de Vos Judith , Bakker Erik NTP , vanBavel Ed
Published: June 2021

Transglutaminase 2 (TG2) is known to be important for both cross‐linking of matrix proteins as well as cell adhesion, cytoskeletal organization and signaling. However, its cellular distribution and mechanisms of secretion and activation during matrix remodeling remain unclear. We tagged TG2 at the C‐terminal with eGFP. TG2/eGFP was transfected into HEK/293T cells using Effectene. Western blot and in vitro activity assay showed a >100‐fold increase in active TG2. No difference was observed in TG2 activity between TG2/eGFP and its non‐fluorescent control.

TG2 distribution in HEK cells strongly depended on the underlying substrate. When seeded on collagen type I, TG2 appeared mainly as intense intracellular and membrane‐bound spots, as well as in microparticles at distances up to 100 micron from the cell. However, on a fibronectin coating, TG2 was rather homogenously distributed throughout the cytosol. In HEK cells plated on plastic, TG2 localized strongly along the cell membrane. Here, transfection induced a large increase in fibronectin staining, which colocalized with TG2 fluorescence. TG2 was almost absent in the nuclei of living cells.

In conclusion, we developed a construct of green fluorescing TG2. HEK cells expressing TG2/eGFP showed distinct localization of TG2, depending on its ECM. Only TG2‐transfected cells were able to produce a membrane‐bound fibronectin matrix.

Supported by NHF grant 2005.B080
Albumingel for cartilage and intervertebral disc regeneration prevents blood vessel formation
Authors: Scholz Beate , Kinzelmann Claudia , Benz Karin , Baumer Yvonne , Mollenhauer Jürgen , Schlosshauer Burkhard
Published: June 2021

Healthy cartilage is devoid of blood vessels, thus an implant matrix for autologous chondrocyte implantation should exhibit antiangiogenic properties. The purpose of the work was to investigate the influence of an albumin gel (AG) for cartilage and intervertebral disc (IVD) regeneration on blood vessel formation. The AG is composed of activated sheep serum albumin, hyaluronic acid and a polyethylene glycol spacer. Primary human endothelial cells (EC) cultured on the AG did not adhere on the surface and did not proliferate. Life‐dead staining displayed an increase in dead cells within 5 days of culture on the AG, while extracts of the AG showed no cytotoxic effect. Controversely, the vast majority of human primary chondrocytes cultured within the AG were vital after 5 days. EC stimulated by vascular endothelial growth factor did hardly invade through AG coated transwell filter. Blood vessels of the chick chorioallantoic membrane did not grow into the implanted AG. Additionally, IVD cells enclosed in the AG were implanted subcutaneously in immunodeficient mice. After 2 weeks there was no evidence of blood vessel formation in the implant.

The results indicate that the AG is a suitable 3D‐matrix for chondrocytes exhibiting nonpermissive properties for EC and therefore could be an ideal implant for cartilage and IVD regeneration.

All procedures were approved by the local Ethics and the local Animal Care and Use Committee.
Rotational Stress Impairs Cutaneous Wound Healing In Mice
Authors: Monte‐Alto‐Costa Andréa , Romana‐Souza Bruna
Published: June 2021

Objective

The aim of this study was to investigate the effects of the rotational stress on cutaneous wound healing in mice.

Methods

Male mice were spun at 45rpm for 15minutes every hour from three days before wounding until euthanasia. Control animals were not submitted to stress. A full‐thickness lesion was created and measured. Fourteen days after, animals were killed and lesions were collected. Sections were stained with HE and immunostained against F4/80 and alpha‐smooth muscle actin to quantify macrophages and myofibroblasts, respectively. Data are expressed as mean±S.E.M and analyzed by Student's t test.

Results

Fourteen days after wounding, wound area was greater in the stress group than in the control group (p<0.05), while re‐epithelialized wound area was smaller in the stress group than in the control group (p<0.05). Stressed mice presented less organized collagenous granulation tissue (rich in inflammatory cells) when compared with control mice. The macrophages number and myofibroblasts density was greater in the stress group than in the control group (p<0.05).

Conclusion

The rotational stress delays the cutaneous wound healing in mice.
3D‐X: making spatial sense of cross‐sectional anatomy in an online learning environment
Authors: Bhattacharyya Siddhartha , Dorosh Kyle , Wilson Tim , Johnson Marjorie
Published: June 2021

Residency programs are demanding an increased knowledge of cross‐sectional anatomy from entering physicians, while medical schools are decreasing time allotted to teaching anatomy. The increasing use of e‐learning resources in medical education led us to create a web‐based tool to facilitate the learning of cross‐sectional anatomy.

A 3D model of the abdomen was constructed in Amira 5.0, by segmenting 600 images from the Visible Male cryo‐section data. A number of cross‐sectional images were imported into Flash CS4 along with five reconstructed models, and interactive controls for the 3D models and dynamic labels for the cross‐sections were programmed using the ActionScript 3.0 engine in CS4. The application, titled 3D‐X, was exported in a web‐friendly flash format and published online.

In 3D‐X, users can view seven key cross‐sections, while the planes of origin are indicated on the 3D model. The 3D model can be rotated and modified to view various structures from different angles. Moving the cursor over an anatomical structure on a 2D cross‐section causes the structure's textual label to appear and colors the structure to match its counterpart on the 3D model. Clicking a structure on a 2D cross‐section modifies the 3D model to create an unobstructed view of the structure and may help users correlate 2D and 3D information.

Based on a research study, 3D‐X co‐administered with cadaveric anatomy improved CT interpretation when compared with learning solely through cadaveric anatomy. 3D‐X therefore shows promise as a tool for enhanced learning of cross‐sectional anatomy by providing a familiar 3D context to aid with spatial orientation.
iMorphology: integrative, blended teaching of anatomy and diagnostic imaging
Authors: Streicher Johannes
Published: June 2021

In integrated curricula anatomy teaching frequently integrates chronologically with other subjects, but fails to integrate with regard to contents; especially, if higher numbers of students shall be taught in small groups. This was also the case in the first approach at the medical university of vienna, where 720 students are enroled per year. After evaluation giving unsatisfactory results, we have now elaborated the iMorphology‐concept for integrated teaching of anatomy and diagnostic imaging. In the same module, students switch between anatomical preparation of an organ system at the dissection hall and according case‐based learning of diagnostic imaging of the respective organ system guided by radiologists. In the dissection hall, ceiling mounted iMacs provide the students with the images from these cases, additional clinical cases, preparation‐videos, videos of relevant surgical/clinical interventions, animations illucidating developmental processes, and 3D‐reconstructions from cross‐sectinal image series. Assessment combines immanent evaluation of dissection and respective background knowledge, mutual cooperative teaching of students among each other, and diagnostic imaging‐tests using interactive learner response devices. With this blending of learning and testing approaches, we achieve faster, indepth and crosslinked understanding of morphology in a yet very limited time frame.
Cadaver Computed Tomography as a Tool for Teaching Anatomy: an Update
Authors: Ellozy Sharif Hamed , Lerner Susan , Reidenberg Joy S. , Weintraub Joshua , Marin Michael L. , Laitman Jeffrey T
Published: June 2021

Cadaver dissection has been the mainstay of anatomy instruction. However, advances in clinical imaging have made familiarity with cross‐sectional anatomy crucial. Additionally, clinical relevance in the preclinical curriculum is key in generating student enthusiasm. In order to facilitate these goals, high resolution computed tomography was performed on all cadavers used in gross anatomy.

Multiplanar and 3D reconstructions were performed on 50 cadavers over two years. Specific reconstructions of skeletal, aerodigestive, and vascular systems were done, as well as any anatomic variants or postsurgical changes. The CT scans were read by a radiologist. Students were given access to all of the studies and instructed on how to use a DICOM viewer. CT images were included on the examinations. Student groups were required to identify a structure of interest in their cadaver, research the clinical significance of this finding, and illustrate the finding with a CT image.

Introducing the principles of imaging early in medical school may facilitate the transfer of knowledge from the dissection laboratory to its clinical application. The ability to identify anatomic variants and pathology a priori allows for focused dissection in select cadavers. We have found cadaver computed tomography to be a valuable addition to traditional dissection in teaching anatomy.
Diagnosis of Pediatric Intussusception using Point of Care Ultrasound in the Pediatric Emergency Department
Authors: Halm Brunhild Minette , Boychuk Rodney B , Franke Adrian A
Published: June 2021

Over the past decade Point‐of‐Care Ultrasound (POC US) is increasingly performed in emergency medicine for a variety of indications. Pediatric emergency medicine physicians have been slower to understand the importance of POC US in the diagnosis of some specific pediatric conditions.

The use of POC US and knowledge of the sonographic characteristics of intussusception can help any emergency physician in the prompt diagnosis of this condition, thereby reducing morbidity that may result from a delay in diagnosis. This report illustrates the use of POC US in a case of a 10 month old male that presented to the emergency department (ED) at midnight with fussiness as the only symptom. A diagnosis of intussusception was made using real‐time POC US by a pediatric emergency medicine attending.

In some pediatric hospitals, like our, the radiologists and the ultrasound technicians are not readily available after hours and need to be called in. The goal is to diagnose and treat intussusception as early as possible since a delay in diagnosis may result in the need for surgical reduction or even intestinal resection. The diagnosis of intussusception is best performed by an experienced sonographer, such as the radiologist. However, if a delay for this study is anticipated POC US by a pediatric emergency physician trained in POC US can be crucial in reducing time to diagnosis and definitive treatment.
Increased Presence of Peer Teaching and Teamwork in Gross Anatomy
Authors: Bernd Paulette , Richards Boyd
Published: June 2021

Gross Anatomy has always included teamwork because students work in groups to accomplish their dissection of the human body. One of the goals of the new curriculum at Columbia University's College of Physicians and Surgeons is to foster teamwork, as well as peer teaching. Several changes were made in the Gross Anatomy course to reflect this mandate. The first change involves student teaching within the Gross Anatomy laboratory. Two groups of four students alternate days in the dissection laboratory. One student from the non‐dissecting group will learn that day's dissection from the dissecting group after which they teach their lab group at the next laboratory session, thereby delegating responsibility to the student. The addition of non‐dissecting sessions that run concurrently with the laboratory is the second change. The non‐dissecting group works in a team to examine bones, prosected materials, radiographic images, surface anatomy and discuss clinical cases. These sessions are written so that they can be done without faculty assistance thereby fostering self‐sufficiency. The third innovation is that the dissecting groups of 4 students take the practical exam together and submit one answer sheet requiring a shared responsibility among students. These changes to the course allow the students to take more responsibility for their education and learn to work with colleagues under conditions that require collaboration and compromise.
Understanding Protein Synthesis: A Role Play Approach in Large Undergraduate Human Anatomy and Physiology Classes
Authors: Sturges Diana , Maurer Trent
Published: June 2021

The study investigated the effectiveness of role play in a large undergraduate science class. The targeted population consisted of 298 students enrolled in two sections of an undergraduate Human Anatomy and Physiology course taught by the same instructor. The section engaged in the role play activity served as a Study group, while the section presented with a traditional lecture served as a Control group. A pre/posttest assessment and a survey were administered to both sections and used in data analysis. In addition, overall test scores and item analysis were examined. The analysis revealed that participants in both groups improved significantly from pretest to posttest, but there was not a significant difference between the groups in the posttest score. Neither group showed a significant change from posttest to the exam. However, there was a moderate positive effect on engagement and satisfaction survey questions from being in the Study group (based on 255 total surveys returned by both groups). Role play activity was at least as effective as the lecture in terms of students' performance on the abovementioned assessments. In addition, it proved successful in engaging students in the learning process and increasing their satisfaction. No research support sources.
Personalizing embryology teaching to increase interest and clinical relevance
Authors: Lyons Virginia
Published: June 2021

Due to its relevance in clinical practice, embryology is an important component of the undergraduate medical curriculum. However, for various reasons, many students do not devote sufficient time and effort to mastering this subject. Some students find embryology difficult to understand, and thus dislike and avoid it; others are unaware of its clinical application so they deem it unimportant. Physicians‐in‐training crave patient contact in any form, thus we hypothesized that personalizing embryology with a patient story would increase student interest and motivation to learn. The story was woven throughout the entire course, and the clinical relevance of the material was emphasized. Initial feedback of this approach has been positive. Quantitative and qualitative data regarding this curricular change will be collected at the end of the term.
Medical gross anatomy laboratory tours have a positive impact on participant interest in the health care professions
Authors: Eckel Christine M. , Pratt Rebecca L , Hermey Donna , Hanna Jandy B
Published: June 2021

To encourage HS and allied health students in West Virginia who have an interest in the health care professions, WVSOM has a laboratory tour program for students enrolled in human anatomy courses at the HS or undergraduate level. Each tour begins with a presentation covering proper laboratory etiquette, the role of the Human Gift Registry, and scientific aspects of cadaver embalming and plastination. Participants are then given a tour of the lab and demonstrations of human anatomy emphasizing clinical applications using cadaveric specimens, radiographs, and/or plastinated specimens. To assess the value of the tours, we ask all participants to complete pre and post tour questionnaires. In the spring of 2009, 255 tour participants returned questionnaires; a response rate of 90.4%. 82% of participants indicated a strong or very strong interest in pursuing a career within the field of health care, and 27% said attending the tour changed their opinion about the health care professions. Of those whose interest changed, 78% indicated they were more interested in a health related career as a result of the tour. We conclude that our anatomy tour program positively influences the interest of participants in the health care professions.

Grant Funding Source: NA
Anatomical dissection: youthful or mature inexperience?
Authors: Ettarh Raj , Perry Gary F
Published: June 2021

Despite the well documented relationship between student profile (race, ethnicity, gender, social status) and the experience of anatomical dissection, evaluation of the influence of age has been limited. Two cohorts of medical students enrolled to a dissection‐based anatomy course were surveyed by Likert questionnaire: a young group (aged 18–24 years) and an older group (aged 22–30 years) admitted to traditional‐entry (school leavers) and graduate‐entry programs in medicine respectively. Incidence of reported dissection‐associated emotional symptoms in young school leavers was significantly lower than in graduate medical students. Common to both cohorts was a tendency toward progressive reduction in apprehension and excitement levels associated with the experience of dissection. Notably, nausea was reported by progressively fewer graduate medical students in contrast to the increased proportion of younger medical students who admitted to being nauseous by their third exposure to dissection. The data suggest that the expected intrinsic adaptability of students to the experience of anatomical dissection is modulated by the age of the learner.
Reaching out to nontraditional students
Authors: Ortiz Phillip
Published: June 2021

Empire State College, as part of the State University of New York, has a proven history of success with nontraditional students. Since our founding in 1971 we have awarded nearly 60,000 liberal arts and professional degrees at the associate, bachelor's and master's levels. Approximately 75% of our students work full time and manage family and community obligations while enrolled. Their average age is 36, and most have earned college credit earlier in their lives. The college's mission is to enable motivated adults, regardless of geography or life circumstance, to design a rigorous, individualized academic program and earn a college degree. As such we have developed a number of progressive and innovative tools and techniques to recruit, retain, and graduate our students. Empire State College faculty are effective mentors who see themselves as ‘guides on the side’, rather than ‘sages upon the stage’, and such a perspective is one that resonates particularly well with nontraditional learners. The focus of this presentation will be on the means by which a dynamic and effective learning environment can be built that promotes the success of students and their faculty. Topics that will be raised include assessment of prior college‐level learning, tutorial and on‐line instruction, and effective mentoring.
A Genome‐wide RNAi screen identifies novel factors involved in the processing of snRNA
Authors: Wagner Eric J , Albrecht Todd R. , Chen Jiandong , Ezzeddine Nader
Published: June 2021

Proper 3′ end formation of snRNA depends on a conserved element termed the 3′ box and is thought to be tightly coupled to transcription. Moreover, there is a poorly understood complex of proteins called the “Integrator Complex” that is thought to mediate the processing of snRNAs. This complex contains at least 12 unique polypeptides that are likely loaded onto RNA polymerase II at the promoter and are involved in 3′ end formation.

We are using the Drosophila U7 snRNA gene as a model substrate to study pre‐snRNA processing. We designed a cell‐based reporter by cloning the U7 snRNA gene upstream of the GFP ORF. In the event that an essential processing factor is depleted, the polymerase will transcribe past the processing site resulting in expression of GFP.

Using this reporter, we performed a genome‐wide RNAi screen in S2 cells to identify factors required for the 3′ end formation of snRNA. We found that nearly all of the Integrator proteins identified biochemically are functionally required for the 3′ end processing of snRNA. Two of these Integrator proteins are indeed required for processing of endogenous U7snRNA in mutant flies and we also found that U4 snRNA biosynthesis is only sensitive to a subset of the Integrator proteins leading to the potential that there are distinct complexes associated at different snRNA genes. We also identified several novel genes that had not been previously implicated in snRNA biogenesis.
Ikaros engages the VPAC1 promoter and recruits HDAC1 in activated HuT78 cells
Authors: Benton Keith D , Hermann Rebecca J , Van der Steen Travis , Failing Jarrett , Dorsam Glenn P
Published: June 2021

Proper regulation of the anti‐proliferative G Protein coupled receptor vasoactive intestinal peptide receptor 1 (VPAC1) is important for normal lymphocyte function. VPAC1 mRNA levels are dependent on the activation status of CD4 T cells, as TCR signaling downregulates VPAC1 expression. Ikaros (IK), a transcription factor critical in proper T cell development has been shown to regulate VPAC1 when ectopically overexpressed in NIH‐3T3 cells. Additionally, the VPAC1 locus contains numerous IK binding sites. IK recruits the chromatin remodeling complex NuRD, a HDAC containing multi‐enzyme complex that represses transcription. We hypothesized that IK recruits the NuRD complex upon activation resulting in its downregulation. Binding of IK and HDAC1 to the VPAC1 promoter and gene was queried using chromatin immunoprecipitation. Both were enriched in the VPAC1 locus in an activation dependent manner in the transformed T cell line, HuT 78. This enrichment correlated with the activation induced downregulation of VPAC1. Future studies will overexpress DNA binding, non DNA binding, and alanine/aspartate mutant IK isoforms to determine their effect on IK binding and HDAC1 recruitment. Engagement of the master regulator of lymphopoesis, IK, at the neuropeptide receptor VPAC1 promoter strengthens the neuroimmunomodulation connection at a molecular level. Funding: NIH‐K01 1K01DK064828 and COBRE 2P20RR05566.
Genetic Evidence for PKCδ Signaling in Thrombin‐Induced NF‐ κB Activation in Endothelial Cells
Authors: Singh Vandana , Thippegowda Prabhakar B , Sundivakkam Premanand C , Vogel Stephen M , Malik Asrar B , Ye Richard D , Tiruppathi Chinnaswamy
Published: June 2021

We have shown recently that Ca2+ entry‐mediated AMPK activation promotes phosphorylation of Ca2+‐independent PKCδ at Thr505 to induce NF‐κB activation in endothelial cells. Here we utilized PKCδ knockout (PKCδ−/−) mice to show that PKCδ is indispensable for protease‐activated receptor‐1 (PAR‐1)‐induced NF‐κB and p38 MAPK activation in endothelial cells. In WT mice, administration of PAR‐1 agonist peptide (TFLLRNPNDK) through jugular vein induced NF‐κB target gene ICAM‐1 expression in lungs, whereas in PKCδ−/− mice, PAR‐1 agonist peptide failed to induce ICAM‐1 expression in lungs. Exposure of wild type (WT) mouse lung endothelial cells (MLECs) to either thrombin or TFLLRNPNDK induced Ca2+ entry, AMPK activation, IκBα phosporylation, nuclear localization of p65/RelA, p38 MAPK phosphorylation, and upregulated ICAM‐1. In PKCδ−/− MLECs, PAR‐1 activation‐induced Ca2+ entry and AMPK activation, but failed to induce IκBα phosporylation, nuclear localization of p65/RelA, p38 MAPK phosphorylation, and upregulation of ICAM‐1. Expression of WT‐PKCδ but not the PKCδ‐T505A mutant in PKCδ−/− MLECs rescued PAR‐1‐induced IκBα phosporylation, nuclear localization of p65/RelA, and p38 MAPK phosphorylation. These results provide unequivocal evidence that Ca2+ entry‐mediated PKCδ activation plays a critical role in the signaling mechanism of thrombin‐induced vascular inflammatory responses.
Histone H4 lysine 20 mono methylation at specific genomic sequences induces transcriptional repression
Authors: Veerappan Chendhore Sai , Congdon Lauren M , Houston Sabrina I , Spektor Tanya M , Rice Judd C
Published: June 2021

Histone modifications play important roles in all eukaryotic DNA‐templated processes. To gain further biological insights into two of these modifications, the mono‐ and trimethylation of histone H4 lysine 20 (H4K20me1 and H4K20me3), ChIP‐chip was performed to identify their precise locations on human chromosomes 21 and 22. Our findings indicate that H4K20me1 is preferentially enriched within specific genes whereas H4K20me3 is preferentially targeted to repetitive elements, strongly suggesting that H4K20me1 and H4K20me3 are physically and functionally distinct histone modifications. Consistent with this, depletion of H4K20me1 resulted in an ~2‐fold increase in the expression of H4K20me1‐associated genes while no changes in expression were observed in H4K20me3‐associated genes. In addition, we found that H4K20me1‐associated DNA sequences were sufficient to nucleate H4K20me1 and induce repression of a reporter gene in vivo. Our results reveal a new transcriptional regulatory pathway whereby the DNA sequences within specific gene bodies can establish H4K20me1 to induce their repression.
Regulation of transcription factor AP‐1 in human fibroblasts in response to cytokines: role in regulation of Matrix Metalloproteinase‐3
Authors: Borghaei Ruth , Chambers Mariah , Gorski Grzegorz , Kirkpatrick Garret
Published: June 2021

Previously we showed that IL‐4 inhibits the IL‐1 induction of MMP‐3, and that there was decreased binding in gelshift assays to a probe containing the MMP‐3 AP‐1 site in the presence of IL‐4. In the present study, the AP‐1 Family TransAm Transcription Factor Assay Kit from Active Motif, as well as chromatin immunoprecipitation studies, are utilized to determine the composition of AP‐1 dimers a)available in fibroblasts and b)actually binding to the endogenous MMP‐3 promoter in the presence of IL‐1 and/or IL‐4. Results show that FosB, Fra‐1, c‐Jun and JunB are all present and capable of binding in fibroblasts stimulated with IL‐1, and that their binding is inhibited by IL‐4. JunD binding is present as well, but is relatively unaffected by these cytokines. Interestingly, there was no increase in cFos binding in response to IL‐1 at the 1 or 3 hour timepoints. Preliminary ChIP experiments using an antibody specific for the active form of c‐Jun, show binding to the endogenous MMP‐3 promoter induced by IL‐1 and inhibited by IL‐4, and this binding correlates with levels of acetylated histone H3 binding the endogenous promoter. Further ChIP experiments will be done with other members of the AP‐1 family. In addition, the signal transduction pathways leading to activation of AP‐1 binding will also be examined. This work is supported by grant R15DE16277 from the NIDCR to R.C. Borghaei.
Use of RNA affinity chromatography to investigate factors involved in selenoprotein synthesis and nonsense decay
Authors: Berry Marla J , Seale Lucia , Gilman Christy , Seyedali Ali
Published: June 2021

Selenocysteine, the 21st amino acid, is the defining component of selenoproteins, a family whose members function in cellular oxidative defenses, male fertility, and thyroid function. Selenium deficiency has been linked to the pathogenesis of cancers, diabetes, cardiovascular disease, neurodegenerative diseases and other diseases in which cumulative oxidative damage is implicated. Selenoprotein mRNAs recode UGA, which typically signals termination, to instead specify insertion of selenocysteine. This recoding process involves assembly of complexes at specific secondary structures in the 3' untranslated regions of these mRNAs termed SECIS elements. When selenium is limiting, UGA is recognized as a termination codon and can trigger nonsense‐mediated decay (NMD) of the corresponding mRNAs. Different selenoprotein mRNAs exhibit differential sensitivity to NMD, and the mechanistic basis for this is not known. We are using glutathione‐RNA (GRNA) chromatography to isolate RNA‐binding proteins that bind selenoprotein mRNAs, to further elucidate the mechanism of selenoprotein synthesis and the factors involved in conferring sensitivity or resistance to NMD. Using GRNA with w.t. and mutant SECIS elements, we have identified three new SECIS‐binding proteins and are characterizing the physiological functions of these proteins in selenoprotein synthesis and NMD.

Supported by NIH grant DK‐47320 to MJB.
Covalent modification of proteins by a small peptide Zfra
Authors: Chang Nan‐Shan , Lee Ming‐Hui , Lin Sing‐Ru
Published: June 2021

Post‐translational modification of proteins is crucial for protein functional maturation and/or degradation. We have previously isolated a 3.5‐kDa protein Zfra. Zfra blocks the function of tumor suppressors WWOX and p53 and inhibit cytochrome c release during the mitochondrial pathway of apoptosis. Notably, upon exposure to phosphate under cell‐ and enzyme‐free conditions, Zfra undergoes a rapid self‐polymerization for more than 50 fold (>200 kDa). When cytosolic proteins undergo “zfration”, the zfrated proteins tend to degrade independently of the ubiquitination/proteasome system. Intratumor injection of a Zfra peptide enhances the growth of skin basal cell carcinoma and breast and prostate cancer cells in nude mice by 3–4 fold in 3 months, suggestive of its inactivation of tumor suppressors (e.g. p53 and WWOX) in vivo. Together, Zfra is a universal modifier of many proteins, and the modification probably occurs independently of enzymes. [supported by NSC and NHRI, Taiwan, and DoD, USA]
Generating Permissive Site‐specific Unnatural Amino Acid Synthetases
Authors: Mehl Ryan A , Miyake‐Stoner Shigeki J , Refakis Christian A.
Published: June 2021

Genetically incorporated unnatural amino acid (UAA) technologies are powerful tools that are greatly enhancing our ability to study and engineer biological systems. To incorporate UAAs into protein, the substrate recognition properties of an aminoacyl‐tRNA synthetase (RS) must be modified. Protocols to do so are technically simple but require time and optimization, which has significantly limited the accessibly of this important technology. At present, engineered RSs are evaluated on their efficiency and fidelity. We propose that a third parameter of substrate recognition – permissivity – is equally important. Permissive RSs, whose relaxed substrate recognition properties allows them to incorporate multiple unnatural amino acids (but not natural amino acids), would eliminate the need to generate new RSs for many new unnatural amino acids. Methods for quickly and easily assessing the permissivity of existing RSs and for generating permissive RSs are presented. We determined the degree of permissivity of two UAA‐RSs for a family of structurally related fluorinated UAAs (19F‐UAAs). We then increased the permissivity of the initial RSs to allow for incorporation of the family of 19F‐UAAs. We expect that results of this work will increase the accessibility of UAA technology and the use of new UAAs in proteins. Supported by F&M funds, NSF‐MCB‐0448297; Research Corporation (CC6364); and ACS‐PRF (42214‐GB4).