bluuebunny/crossref_metadata_2025 huggingface dataset:
Hedonics and the cephalic phase fat response in humans
Authors: Mattes Richard David
Published: June 2021

This study tested the hypothesis that the cephalic phase fat response would be greater after oral exposure to a more palatable high fat stimulus. To‐date, 13 adults have been randomized in a cross‐over trial where they received, in random order, oral exposure to full‐fat or non‐fat cream cheese alone, or full fat cream cheese containing either 22.2%w/w sucrose or 3.6%w/w NaCl. On test days, held one week apart, participants were catheterized and then ingested 50g of safflower oil in capsules, to preclude oral fat exposure, followed by 10s of modified‐sham feeding of 5.2g portions of the day's stimulus at 30second intervals for 15 minutes. Blood samples were collected at minutes ‐15, 0, 10, 20, 30, 40, 50, 60, 120, 240, 360 and were analyzed for plasma TAG. The sweet stimulus was rated as the most palatable stimulus. It also led to greater peak plasma TAG concentrations during the first phase response (0‐60m) as well as over minutes 60‐360. These data suggest that adding a palatable concentration of sweetener to a high fat stimulus augments the cephalic phase and later post‐prandial plasma TAG concentration. Whether this is a sweetener or palatability effect requires further exploration. Supported by: NIH R01 DK 0456294.
Slit2‐Robo1 regulates control of SDF‐1 induced T cell adhesion in NOD mice
Authors: Glawe John , Davis William , Kevil Christopher G
Published: June 2021

We have recently reported that SDF‐1/CXCL12 paradoxically attenuates diabetogenic NOD/LtJ T cell firm adhesion to TNF‐α stimulated islet microvascular endothelial cells; however, the reason for this response is unknown. Here we report that NOD/LtJ T cell adhesion responses to SDF‐1 are modulated by Slit2‐Robo1 signaling, with increased Slit2‐Robo1 binding causing reduced adhesion in response to SDF‐1. We investigated whether differing basal levels of Slit2 or Robo1 are responsible for the diametric effects of SDF‐1 concentration on adhesion of NOD/LtJ and C57BL/6J mouse T cells. Western blot and flow cytometry analysis shows that Robo1 expression is significantly elevated in NOD/LtJ mice compared to C57BL/6J mice. Blocking Slit2‐Robo1 interactions with an anti‐Robo1 antibody in parallel plate flow chamber adhesion assays shifted the effect of SDF‐1 from chemorepulsion to chemoattraction clearly implicating Robo‐1 in regulating SDF‐1 adhesion responses. These results demonstrate that autoimmune diabetogenic T cell recruitment and adhesion is highly dependent on Robo1 expression and identifies a previously unknown mechanism of regulating chemokine dependent leukocyte recruitment.
Dysregulation of MicroRNAs Drives the Expression of a Hypermethylation Defect in Breast Cancer
Authors: Sandhu Rupninder , Roll J Devon , Coleman William B
Published: June 2021

A subset of breast cancers express a hypermethylation defect characterized by DNMT hyperactivity, overexpression of DNMT3b, and concurrent silencing of numerous genes. This hypermethylation defect is a feature of most basal breast cancers. Our goal was to determine the mechanism responsible for DNMT3b overexpression in model breast cancer cell lines. Several microRNAs (miRs) have been implicated in the regulation of DNMT3b. To investigate the role of miRs in DNMT3b overexpression, we analyzed miRs‐29a, 29b, 29c, 148a, and 148b in a panel of 16 breast cancer cell lines classified as hypermethylators (n=10) or low frequency methylators (n=6). 80% of hypermethylator cell lines express low levels of >3 miRs and 83% of low frequency methylator cell lines express >3 miRs at normal levels. Cell lines with high DNMT3b levels (SUM185, SUM102, SUM149) expressed all miRs at low or negligible levels, whereas cell lines with low DNMT3b levels (MDA‐MB‐415, MDA‐MB‐468, BT20, ZR‐75‐1) expressed all miRs normally. Further, miR expression patterns correlated with methylation‐sensitive gene expression among these cell lines (R=0.54, p=0.012). These findings suggest that dysregulation of miR expression drives DNMT3b overexpression in hypermethylator cell lines, and identifies DNMT3b and its regulatory miRs as targets for development of novel therapeutic strategies for treating basal breast cancers.

Support: NIH CA78343
The Role of microRNAs in Liver Regeneration in Rats
Authors: Paranjpe Shirish , Bowen William , Michalopoulos George K , LaFramboise William A
Published: June 2021

The discovery of endogenous small (18 ‐ 25 nucleotide) noncoding microRNAs (miRNA) as powerful negative regulators of gene expression has had an enormous impact in biology. It has been estimated that miRNAs regulate about 30% of genes in humans, with an average of 200 mRNA targets/microRNA. Liver regeneration after a two‐thirds partial hepatectomy (PHx) is an extremely complex process requiring interaction and cooperation of many growth factors and cytokines and crosstalk between multiple pathways. To our knowledge, not much is known about the role of miRNAs in liver regeneration in rats following a PHx. MiRNA profiling in regenerating rat liver at various time points post PHx, post sham operation and resting rat liver was carried out. Microarray data of profile obtained at one‐hour was analyzed by ANOVA to test for independent effects of time versus PHx and control groups for differentially expressed miRNA. Preliminary analysis of microRNA profile in control and regenerating liver indicated that 28 miRNAs were significantly regulated in the PHx group as compared to resting liver and sham control. The miRNAs of let‐7 family were significantly down regulated and was confirmed by RT‐PCR. In conclusion, miRNAs predicted to target genes associated with cellular proliferation, transcription factors and apoptosis were significantly regulated with respect to both class (sham operated controls and PHx) and time post PHx.
Minority Faculty Decisions: From Teaching to Administration to Moving Outside of Academia
Authors: Jordan Tuajuanda C
Published: June 2021

Due to a variety of reasons, the number of women and underserved minorities obtaining Ph.D.s has increased over the past 20 years. There has been a concomitant, although not as substantial, increase in the numbers that enter the academy as faculty and pursue careers in research and/or teaching. At many institutions, the tendency is to identify faculty who exhibit excellence in both leadership and research and/or teaching and groom them to enter positions in academic administration. What could be the long‐term impact of this policy on the numbers of minority scientists pursuing academic careers? Can this policy affect the numbers who persist through the tenure and promotion processes? Is movement from the classroom and laboratory to an office good for the professional career of the faculty member? This presentation will touch on these issues in the context of one woman's path from academic teaching and research to university administration and beyond.
Transcript elongation mechanisms
Authors: Svejstrup Jesper Q , Aygün Ozan , Sigurdsson Stefan , Saeki Hideaki
Published: June 2021

Transcript elongation by RNA polymerase II is a surprisingly complex process that is affected by a number of different factors. Biochemical analyses of transcription support the idea that transcript elongation involves frequent pausing and even arrest, which has to be overcome in order for overall transcription to be efficient. A defining response to any transcription obstacle is retrograde motion of RNAPII, so‐called backtracking, during which RNAPII looses contact with the 3′‐end of the RNA and often needs the help of general elongation factor TFIIS (also called SII) to recover. However, a large number of factors associate with, and affect the function of elongating RNAPII. In this talk, our recent studies of the basic mechanism of transcript elongation, including the effect of such co‐factors, will be discussed.
Calcium dependent exocytosis and endocytosis: novel trafficking pathways revealed by the study of host/pathogen interactions
Authors: Andrews Norma
Published: June 2021

Elevation in cytosolic Ca2+ triggers exocytosis of lysosomes, a process required for trypanosome invasion (1) and the resealing of membrane wounds (2). Interactions of bacteria with host cells also involve activation of the plasma membrane repair response. Permeabilization of phagosome membranes by the type three secretion system of the bacterial pathogen Salmonella triggers early lysosomal fusion and bacterial killing (3). SLO, a bacterial pore‐forming toxin, also triggers Ca2+ dependent lysosomal exocytosis and plasma membrane repair. However, the process responsible for removal of the toxin pores from the plasma membrane is a novel, rapid form of endocytosis that follows the exocytosis of lysosomes (4). Characterization of this injury‐induced form of endocytosis showed that it is triggered by the release of specific lysosomal hydrolases. These new findings demonstrate that calcium dependent exocytosis of lysosomes and endocytosis are functionally linked.

This work was supported by the NIH.
A novel gene expression system using transcription amplification to examine cdk2‐associated cell cycle regulator role in cancer cell apoptosis
Authors: Figueiredo Marxa L , Sato Makoto , Wu Lily
Published: June 2021

In order to improve understanding of the contribution of p27kip1, a cyclin‐dependent kinase 2 (cdk2) regulator to growth inhibition in prostate cancer cells, we have modulated p27 expression using a new two‐step transcription amplification system. p27 expression usually is associated with G1/S arrest through cdk2 inhibition, and downregulated during prostate cancer progression by its increased targeting for ubiquitin proteasome degradation. We selected a degradation‐resistant mutant (T187M/P188I;p27mt), hypothesized to be more effective in inhibiting tumor growth. We adapted an adenoviral‐based transcriptional amplification system containing a prostate‐specific promoter (PSE‐BC) driving a GAL4‐VP16 activator, which in turn activates expression of a bidirectional Luc/p27 (or p27mt) cassette. p27mt showed higher growth inhibition, with ~80% tumor growth reduction in vivo, and the main mechanism of growth inhibition was through apoptosis induction. Using a qPCR array, we observed several proapoptotic gene expression changes, including death receptors/ligands, caspases, and TP73 upregulation. Prosurvival changes also were observed, for example, Birc8 upregulation. Further optimization of this system is in progress and includes combination vectors for enhanced antitumor effectiveness, including cell cycle regulators (p12/cdk2ap1) and antisurvival strategies (shHer2).
Reduced Rap1 Signaling Is Associated with Prostate Cancer Progression, Migration, Invasion, and Metastasis
Authors: Henderson Veronica Mwihaki , Osunkoya Adeboye , Zhou Wei , Williams Daron , Anderson Noelani , Kitayama Hitoshi , Csete Marie , Moreno Carlos Sanchez
Published: June 2021

Rap1 is a small G‐protein regulator of E‐cadherin cell‐cell junctions and integrin‐mediated cell‐matrix adhesions. Our expression profiling of clinical prostate cancer tissue samples showed significantly decreased RAP1A mRNA levels in tumor compared to normal tissue samples, while a negative regulator of RAP1, RAP1GAP, was significantly increased. Furthermore, these changes in RAP1A and RAP1GAP expression are significantly correlated with Gleason tumor grade, suggesting that reduced RAP1 signals play a role in metastatic progression. We have analyzed RAP1GAP protein expression by immunohistochemistry using a tissue microarray with 139 cores from 79 prostate cancer patients, and observed that RAP1GAP staining intensity was significantly correlated with tumor grade (p < 0.001). We also show that constitutively active Rap1 signaling slows prostate cancer cell migration and invasion in vitro, while inhibition of Rap1 signaling accelerates cell migration and matrigel invasion. The PTEN‐PI3K pathway interacts with RAP1 signaling, but the mechanism is not well understood. Here we present data showing that the PTEN‐PI3K pathway is an important regulator of RAP1A and RAP1B gene expression.
EST‐based gene silencing for identification of macrophage resistance mechanisms against Francisella tularensis
Authors: Sendzik Tanja , Quan Lu , Kobzik Les
Published: June 2021

Francisella tularensis (FT) is a gram‐negative, highly infectious, facultative intracellular pathogen that causes tularemia, and is a potential bioterrorism agent. Macrophages represent the main site of initial FT replication but the mechanism of survival and bacteriostasis upon activation remains poorly characterized. We used genome‐wide genetic screen to identify host genes responsible for survival of infection. We used macrophages (RAW 264.7) transduced with an EST‐lentiviral "knock‐down" library targeting 40,000 sequences. The macrophages were infected with attenuated Live Vaccine Strain (LVS) FT expressing GFP. We screened for EST interference resulting in a phenotype that allows survival of infection and intracellular bacterial proliferation. Flow cytometric sorting of cells with highest FT load (i.e. top 1% by green fluorescence) was performed. The EST inserts from sorted cells were amplified from genomic DNA by PCR, and corresponding EST inserts were identified by cloning and DNA sequencing. Initial screening has analyzed 1400 clones from 6 different sorts and has identified 7 genes of interest based on repeated detection. Validation of potential targets is underway to confirm gene expression and to assess effects of directed lentiviral knockdown of candidate genes. This approach may prove useful to identify mechanisms in macrophage innate immunity against intracellular pathogens.
µ‐Calpain promotes epithelial cell survival and adherens junction disruption during Helicobacter pylori infection
Authors: O'Connor Pamela M , Lapointe Tamia K. , Buret Andre G.
Published: June 2021

H.pylori (Hp) is a risk factor for gastritis, gastric ulcers, and adenocarcinoma. The ability of Hp to disrupt intercellular adhesion and alter cell survival may influence the clinical outcome of infection. Calpain family proteases cleave the adherens junction (AJ) components E‐cadherin and β‐catenin, and modulate pathways regulating cell survival and apoptosis.

AIMS

1) to assess the effects of Hp on calpains 2) to assess the effects of calpains on Hp‐induced AJ defects and cell survival.

RESULTS

Epithelial (MKN45 and SCBN) monolayers were challenged for 24h with Hp strain SS1 or the human isolate 60190 in the presence or absence of calpain inhibitor PD150606. Immunoblotting revealed that Hp‐challenged monolayers displayed elevated levels of active µ‐calpain and the cleaved form of the calpain substrate α‐spectrin. Immunoblotting and immunostaining revealed calpain‐dependent, Hp‐induced E‐cadherin redistribution and cleavage of the 120kDa full‐length E‐cadherin to a functionally‐impaired 100kDa form. Also, Hp induced membrane‐to‐nucleus translocation of β‐catenin in a calpain dependent fashion. Finally, calpain inhibition enhanced Hp‐induced apoptosis, PARP cleavage, and caspase‐3 activation.

CONCLUSION

Hp‐induced activation of µ‐calpain mediates processes associated with tumorigenesis including AJ disruption and promotion of abnormal cell survival. Funded by CIHR.
Active learning in a large classroom held in a conventional auditorium
Authors: Bailey Cheryl , Matejka Stephanie , Thompson Kyle
Published: June 2021

Our long‐term goal is to educate biochemistry students about how scientific knowledge is created. An immediate challenge is to engage students while covering a large amount of material with >120 undergraduate students seated in a large auditorium. We introduce student‐centered learning and opportunities to practice biochemistry in a conventional large class setting. Active learning through POGIL (process‐oriented guided‐inquiry learning) and a semester‐long genome annotation project are combined with opportunities to practice biochemistry through online and in‐class strategies. On‐line and in‐class practice includes online quizzes, online group discussion, online assignments, in‐class quizzes, in‐class clicker questions, and extensive study questions. Student surveys assess student perceptions of the opportunity to practice biochemistry multiple times throughout the semester. This allows for active engagement in class, and to work in teams to interpret models, build concepts, and test these concepts in new situations.
Utilizing Students' Experience with Cancer to Teach Biochemical Concepts to Non‐Science Majors
Authors: Kruchten Anne E
Published: June 2021

At Linfield College, all students are required to complete course work in the category "Natural World" as part of the broad liberal arts curriculum. Non‐science majors are encouraged to take courses specifically designed to train a science‐literate public. BIOL 109: The Life and Death of Cancer was designed to utilize students' preexisting interest in cancer, stemming mostly from personal interaction with the disease, to train them to be conversant in the language and ideas of science. During the course, the students learned fundamental biochemical concepts such as replication, transcription, and translation, as well as advanced concepts such as apoptosis and cellular transformation. A pre course exam and a post course exam identified the extent of learning, and specific assignments taught them to apply their knowledge of science to the economic, political, and social impacts of cancer. Post course evaluations demonstrated that students were enthusiastic about understanding science and now felt more inclined to initiate discussions with their peers and others. This course will continue to be offered to stimulate general public interest in scientific concepts.
Amniotic Fluid‐derived Stem Cells For Regeneration of Infracted Rat Myocardium
Authors: Guan Xuan , Delo Dawn , Wang Zhan , Shaffer Robyn , Groban Leanne , Callahan Michael , Smith Tom , D'Agostino Ralph B. , Atala Anthony , Soker Shay
Published: June 2021

Objective

This experiment was set to determine if human amniotic fluid‐derived stem (hAFS) cells could be differentiated into cardiac lineage, engraft into infarcted myocardium and affect heart function.

Methods

AFS cells were treated with 5‐aza‐2‐deoxycytidine and analyzed for expression of cardiac markers by PCR and immunostaining. For in vivo study, 5 million cells were injected into hearts immediately after infarction. Heart function was monitered by echocardiography and invasive hemodynamic study. All hearts were analyzed 3 months after injection.

Results

Differentiated cells expressed cardiac Troponin I, Troponin T, Myosin light chain 2v, MEF2C, MEF2D and Nkx2.5. Undifferentiated and differentiated hAFS cells engraft into infarcted hearts. When compared to control, injection of undifferentiated cells reduced left ventricular end systolic diameter (p=0.098) and improving fractional shortening (p=0.074) while differentiated cells had opposite effect (p = 0.017 and 0.01).

Conclusions

The result indicates that hAFS cells can be differentiated into cardiac phenotype, engraft into infarcted myocardium and affecting cardiac function. Undifferentiated hAFS cells tend to improve cardiac function while differentiated cells worsen function. The reason for this difference remains unclear.
Effects of nicotine on avian neural crest migration and embryonic development
Authors: Brauer Philip R. , Reedy Mark V. , Smith Derek David , Olley John
Published: June 2021

The types of congenital defects associated with smoking include organs dependent on the formation and migration of neural crest (NC) cells. Studies show high levels of nicotine can induce neural tube defects and restrict growth. However, virtually nothing is known regarding its effect on NC morphogenesis or development of NC‐dependent organs. Here, we tested whether nicotine, at concentrations found in human amniotic fluid, increased the risk of neurocristopathies in avian embryos and if it altered NC cell migration and attachment. We found a single dose of nicotine (100 nM) given at the gastrulation stage in ovo increased the frequency of neural tube defects, reduced embryonic wet weight, and delayed developmental progression. Moreover, the early cranial and cardiac NC migration pattern was perturbed. In vitro, NC cells obtained from nicotine‐treated embryos also exhibited altered cell migratory behavior and cell attachment. Collectively, these studies show even a single exposure to a low dose of nicotine is detrimental to early NC morphogenesis and embryonic development. Supported by Nebraska Health and Human Services (LB506) and NIH (#P20‐RR0169).
Design of interactive computer module for the study of histology
Authors: Prensky Colin J. , Mak Ki M. , Shaw Phyllis A. , Reidenberg Joy S.
Published: June 2021

This project developed a model of a digital study tool for learning histology that is keyed directly to the Mount Sinai Histology Slide Collection (MSHSC). This ensured that the visual appearance of various structures would correlate directly between the illustrations and slides available to students. The developed module focused on H&E stained tissues from the small intestine. Digital photographs were captured directly from the MSHSC using a Nikon Eclipse 50i microscope. Two‐dimensional digital drawings were traced over these images using Adobe Illustrator and Photoshop. Composite illustrations (photos and drawings) were created at low and high magnifications. These drawings were then imported into Adobe Flash in order to create a simple user interface that allows students to interact with the illustrations. The low magnification highlighted the tissue layers, while high magnification emphasized cellular components (e.g., brush border). The interface allows the student to navigate between different magnification views of the tissue section as well as highlight various tissue layers, cell types, and specific cell features by clicking on buttons from a list of key structures. The template developed in creating this digital study aid can be used for further expansion of the project into a comprehensive series that will include modules illustrating the entire series of the MSHSC.

Grant Funding Source

Mount Sinai School of Medicine
Techniques of Developing Illustrations for Histology Teaching
Authors: Cui Dongmei , Lynch James C , Yang Gongchao , Haines Duane E
Published: June 2021

Illustrations are essential aids in teaching histology and other anatomy courses. They can be particularly helpful when they are specifically customized to fit exactly with the material in a course. Instructors in our department have extensively used CorelDraw to develop illustrations for lectures, lab manuals, textbooks, and test questions. This program allows illustrations to be developed in several layers. Typical steps include: (1) present basic ideas in an outline layer; (2) provide colors in background layers; (3) add colors to drawing in fill layer; (4) add details; (5) indicate details with leader lines or arrows; and (6) label with text. Completing the illustrations in separate layers gives us more flexibility, such as adding a new layer or blocking a layer which may be used at another time. One particular advantage in this approach is the opportunity for faculty to develop and use, in their own courses, high quality artwork that is comparable to that which is commercially available. The cost of developing an illustration is less than when a professional artist is employed, and the instructor's better understanding of cells, structures and tissues results in better illustrations. In summary, creating illustrations by instructors for teaching histology or other courses is efficient, practical and economical, especially in times of tight school budgets.
Clinical relevance of cadaver dissection of the root of the neck
Authors: Hankin Mark H , Stoller Jeremy
Published: June 2021

An essential issue in anatomy education is to determine the method of teaching that produces the most effective clinicians (McLachlan & Patten, 2006). One question is whether students can apply knowledge gained through dissection in a clinical setting? To address this we designed an experiment to test whether a modified dissection of the root of the neck can help students remember, understand, and apply anatomy relevant to clinical procedures such as subclavian and internal jugular venous cannulation or brachial plexus blocks. The modified dissection involved complete removal of the clavicle by disarticulating the SC and AC joints, rather than cutting it at its midlength and leaving much of it in place. When performed with care, this results in the clear exposure of structures such as subclavian vessels, brachial plexus, and phrenic nerve. We plan to test our question using assessment at three cognitive levels: (1) Remember [e.g., Identify the structure]; (2) Understand [e.g., Which nerve(s) could be stimulated by a needle inserted into the skin over the internal jugular vein?] and (3) Apply [e.g., On an undissected cadaver, show the position of the entry site at which you would introduce a needle to start a subclavian venous line.].
The Uses of Soft Embalming for Cadaver‐Based Dissection, Instruction in Gross Anatomy, and Training of Physicians
Authors: Boaz Noel T , Anderhuber Friedrich
Published: June 2021

Soft embalming (Thiel, W. Annals of Anatomy (1992) 174:185‐195; (2002) 184:267‐269) is a technique which relies on a mixture of salt compounds and very low amounts of volatile formaldehyde and formalin to effect fixation of tissue with a number of unique properties. Cadavers preserved with the Thiel Method have no detectable odor, a lifelike flexibility of body parts, excellent color preservation of muscle, viscera, and vasculature, and superior antimicrobial preservation properties. Introduction of embalming fluid through the external iliac artery and drainage from the superior sagittal sinus spares the inferior neck and femoral triangle regions from damage during the embalming procedure. This procedure presents a number of significant didactic advantages over traditional methods of embalming. Because of the more lifelike texture and color of structures students' dissections are of high quality, improving learning in the laboratory. Appreciating the actions of muscles at joints, especially in the extremities, is facilitated by the flexibility of the cadaver. Soft‐embalmed cadavers offer advantages over unembalmed cadavers or simulators in teaching clinical procedures, such as intubation, lumbar puncture, central line placement, thoracocentesis, and all fields of surgery, including pelvic floor repair and face lift. The flexibility of the peritoneal membrane and internal viscera allows soft‐embalmed cadavers to be especially useful in teaching laparoscopic surgical anatomy. Soft embalming holds much promise for further innovative educational uses in cadaver‐based anatomical and clinical instruction.
Teaching the clinical anatomy of the paranasal sinuses using 3‐dimensional cone beam computed tomography (CBCT) imaging in dentistry
Authors: Smith Kyle D. , Lanphier Terry F. , Saini Tarnjit S. , Jergenson Margaret A. , Norton Neil S.
Published: June 2021

The paranasal sinuses are invaginations from the nasal cavity that drain into spaces associated with the lateral nasal wall. Each sinus (frontal, maxillary, ethmoidal, and sphenoid) is named after the cranial bone where it is located and is lined by a respiratory epithelium. The anatomy of the paranasal sinuses has not typically been used as a diagnostic tool in general dentistry, but there are examples of its use in certain specialties. Most cephalometrics have concentrated on areas such as orthodontic treatment, and cranial growth and development in the youth. However, the introduction of cone beam computed tomography (CBCT) provides 3‐dimensional imaging at sub‐millimeter resolution that may serve as an adjunct to pre and post‐operative treatment. For many general dentists, the paranasal sinuses have often been neglected. However, the sinuses are subject to many pathologies which can be observed using CBCT. For example, sinusitis (especially maxillary), polyps, and osteomyelitis are readily seen. Additionally, the use of implants, both maxillary and mandibular, are becoming more common. Thus, it is imperative for the general dentist to have a thorough understanding of the normal anatomy of the paranasal sinuses. In this study, we sought to use CBCT to 3‐dimensionally to aid in the instruction of the clinical anatomy of the paranasal sinuses.

Grant Funding Source

Departmental Funds
Clinico‐Histologic Conferences (CHC): A New Endeavor in Teaching Histology
Authors: Shaw Phyllis A. , Friedman Erica
Published: June 2021

Clinico‐Histologic Conferences were developed for the histology course in the first year of medical school to: enhance the relevance of histology by providing a clinical context, foster peer teaching and learning through teamwork, and promote longitudinal integration and application of medical knowledge. Twenty‐four groups of six students each were assigned a disease and created a 10 min presentation with a handout with the objective of teaching the class about that disease. The students' oral presentation and handout focused on the salient features of the disease, i.e. description of the disease, including symptoms, findings, incidence (US & other countries), age or gender or other predisposing factors and outcomes (morbidity, life expectancy); etiology of the disease (cause or origin of the disease, the basis of the cellular manifestation of the disease, and if known, the molecular/genetic defect.); diagnosis (lab tests, histological tests). A 5 min Q and A was at the end of each presentation with an expert MD facilitator present. The facilitator evaluated the students on their oral presentation (choice of content, format, and discussion to facilitate student understanding of the disease) and handouts (quality of information, format, clarity of information and graphics of histology/pathology of disease). There were also questions on the midterm and final exams related to the content taught in the CHCs.
Cell cycle‐dependent binding of replication and transcription proteins to the human DBF4 ori
Authors: Romero Julia , Lee Hoyun
Published: June 2021

We have previously identified an origin of DNA replication (ori) at the transcriptional promoter of the human DBF4 gene. Using a novel replication initiation point (RIP) mapping procedure, we have shown that replication from the DBF4 ori initiates from multiple potential start‐sites which are distributed within two zones separated by ~400‐bp on the complementary DNA strands. Initiation from the two replication zones occurs sequentially, which we termed asymmetric bidirectional replication (ABR). Using chromatin immunoprecipitation (ChIP) assays we have studied protein‐DNA interactions at the DBF4 ori during cell cycle progression. Our data show that the origin recognition complex (ORC) binds to both replication zones simultaneously during late G1. However, during the G1/S transition and following S‐phase entry, ORC is sequentially released from its binding sites, mirroring the ABR pattern. ChIP data also suggests that binding of the Sp1 transcription factor and replication proteins to the DBF4 locus is mutually exclusive. This data is consistent with the idea that transcription and replication are inversely correlated at the DBF4 locus. Overall, this study provides new insight into how DNA replication and transcriptional activities are regulated in a concerted manner at ori/promoters in mammalian cells. This work was supported by grants from the Canadian Institutes of Health Research to H.L.
High‐resolution analysis of DNaseI‐hypersensitive sites in a muscular dystrophy‐linked subtelomeric region of 4q
Authors: Xu Xueqing , Tsumagari Koji , Crawford Greg , Ehrlich Melanie
Published: June 2021

DNaseI hypersensitive (DH) sites are associated with nucleosome‐free chromatin subregions often in promoters, enhancers, and locus control regions. We used DNase‐chip to examine DH sites in two regions of special interest to facioscapulohumeral muscular dystrophy (FSHD). This dominant disease remains enigmatic even after five expression microarray studies. Custom tiling arrays for this study represented 4q35.2, including D4Z4 arrays of tandem 3.3‐kb repeats whose contraction is linked to FSHD, and 10q26.3 with its almost identical, but phenotypically neutral, D4Z4 arrays. In the center of the 4‐Mb 4q35.2, there was a low density of DH sites, consistent with its low density of known genes. Some 4q35.2 or 10q26.3 genes in three FSHD and three control myoblast cell populations had DH sites just in 5'gene regions (e.g., FRG1), and others had DH sites at many intragenic positions (e.g., FAT and INPP5A). DH sites were observed in sequences corresponding to hypothetical proteins (C10orf92 and C10orf93) and in subregions far from known genes. The 4q‐specific nature of FSHD is likely to be due to differences between the chromatin landscape of 4q35.2 and 10q26.3 that make only 4q D4Z4 contractions pathogenic. Our data illustrate the large‐scale differences in chromatin in these subtelomeric regions and suggest where to look for FSHD‐relevant long‐distance chromatin interactions. (Supported in part by NIH Grant NS048859)
A canonical promoter organization of the transcription machinery and its regulators in the Saccharomyces genome
Authors: Venters Bryan J , Pugh B. Franklin
Published: June 2021

The Pol II transcription machinery assembles and operates in the context of nucleosomes, which have well‐defined canonical positions at the beginning and end of protein‐coding genes. However, the predominant organizational theme by which the transcription machinery and chromatin regulators are positioned within promoter regions or throughout genes in a genome is largely unknown. We have mapped the genomic location of diverse representative components of the gene regulatory machinery in Saccharomyces cerevisiae to an experimental resolution of <40 bp using ChIP‐chip. Defining how these factors are arranged with respect to each other and to promoter nucleosome positions should provide insight into how the organization of the transcription machinery at promoters regulates gene expression. We find that the ‐1 nucleosome, which establishes the upstream border of the promoter region, is a likely target of substantial regulation (assuming that occupancy is linked to function) since nearly all tested chromatin remodeling complexes selectively occupy the ‐1 nucleosomal region compared to the +1 region. Furthermore, we find that the ‐1 nucleosome is depleted rather than laterally displaced when Pol II is present, but not when only TBP and TFIIB are recruited. This suggests that the ‐1 nucleosome is removed during PIC assembly, after TBP and TFIIB have been recruited, but before or concurrent with Pol II recruitment, which might explain the prevalence of remodeling complexes in the vicinity of the ‐1 nucleosome.

This work was supported by NIH grant ES013768.
Up‐regulation of human iNOS transcription by p300 and AP‐1 interaction
Authors: Guo Zhong , Shao Lifang , Geller David A
Published: June 2021

p300, a ubiquitous transcription coactivator, plays an important role in gene activation. Our previous work demonstrated that human iNOS (hiNOS) expression can be highly induced with the cytokine mixture (CM) of TNFα + IL‐1β + IFNγ. In this study, we investigated the functional role of p300 in the regulation of hiNOS gene expression. Our initial data showed that overexpression of p300 significantly increased the basal and cytokine‐induced hiNOS promoter activities in A549 cells. Interestingly, p300 activated cytokine‐induced hiNOS transcriptional activity was completely abrogated by deleting the upstream hiNOS enhancer at ‐5 kb and ‐6 kb in the promoter. Furthermore, p300 over‐expression increased cytokine‐induced transcription activity on a heterologous minimal TK promoter with the same hiNOS enhancer. Site‐directed mutagenesis of the hiNOS AP‐1 motifs revealed that an intact upstream (‐5.3kb) AP‐1 binding site was critical for p300 mediated cytokine‐induced hiNOS transcription. Lastly, our ChIP analysis and gel shift assay proved that p300 was binding to Jun B proteins at ‐5.3 kb AP‐1 binding site in vivo and in vitro. Our results suggest that coactivator p300 mediates cytokine‐induced hiNOS transactivation.
Physical Models of transcription factors activated via histidine kinase two‐component signal transduction signaling pathways
Authors: O'Mara Deirdre E , Stock Ann , Herman Tim , Feng Ashley , Hanchuk Stephanie , Oliveira Vitor , Kolb Catherine , Wei Victoria , Young Andrew , Contess Susan
Published: June 2021

The 2009 Pingry S.M.A.R.T. Team (Students Modeling A Research Topic) has been working with Ann Stock of the Center for Advanced Biotechnology and Medicine at UMDNJ ‐ R .W. Johnson Medical School to design and produce accurate, physical models of transcription factors activated via histidine kinase two‐component signal transduction signaling pathways using rapid prototyping (RP) technology. Two‐component signal transduction, based on phosphotransfer between a histidine protein kinase and response regulator protein, is the most prevalent multi‐step signaling strategy in bacteria. The project focused on the conserved regulatory and effector domains in response regulator proteins and the conformational changes that accompany phosphorylation‐induced activation. Discussions with the Stock lab allowed students, using RP‐RasMol to design models of E. coli PhoB and M. tuberculosis MtrA to highlight structural and functional characteristics. The physical models which serve as "communication tools" used to enhance the understanding and applications among the scientific community. Contributing this tool to the Stock laboratory, the students have had unique opportunity to experience and participate in the activities of a research laboratory. Work is supported by grant awarded to T.Herman by the NIH NCRR SEPA and HHMI.
Nitric Oxide Enhances NFATc‐mediated Aquaporin‐2 Expression
Authors: Gonzalez Bosc Laura V , de Frutos Sergio , Kanagy Nancy L , Majowicz Mónica P , Vidal Norberto A , Albertoni Borghese María F
Published: June 2021

The Ca2+/calcineurin regulated transcription factor, NFATc, has been shown to regulate aquaporin‐2 (AQP2) expression in renal collecting duct cells. Nitric oxide (NO) it is also implicated in AQP2 regulation. Therefore, the goal of this study was to investigate whether NO affects NFATc regulation of AQP2 promoter. MDCK cells were cotransfected with an AQP2 promoter luciferase reporter and a renilla luciferase plasmid. Cells were treated for 24 h with 1 μM ionomycin, (Io, calcium ionophore, NFATc activator), 100 nM phorbol 12‐myristate 13‐acetate (PMA, PKC\AP‐1 activator) in the presence or absence of 0.1 mM NONOate (NO donor) or 1μM cyclosporin A (CsA, calcineurin inhibitor) or 10μM ODQ (soluble guanylyl cyclase inhibitor). AQP2 promoter activity increased in cells treated with Io+PMA (2.1±0.1* fold from control). Incubation with NONOate in addition to Io+PMA produced a further increase in promoter activity (2.8±0.2*&) that was attenuated by CsA (1.9±0.1*#) and ODQ (2.0±0.1*#). *p<0.05 vs. control, #p<0.05 vs. NO+Io+PMA, &p<0.05 vs. Io+PMA. NONOate did not have any effect by itself. Similar results were observed in cells transfected with a 9xNFAT‐luciferase reporter demonstrating that NFAT activity is enhanced by NO. Our data suggest that NO via cGMP has a synergic effect with Io+PMA in activating the AQP2 promoter in MDCK cells. NO could be enhancing NFATc nuclear import or reducing its export to enhance NFATc‐mediated AQP2 promoter activation. Supported by AHA SDG and HL088151.
Inhibition of gene external RNA polymerase III transcription by PTEN
Authors: Cabarcas Stephanie M , Schramm Laura
Published: June 2021

RNA polymerase III is responsible for transcribing many of the small structural RNA molecules involved in RNA processing and protein translation, thereby regulating the growth rate of a cell. Proper initiation by RNA polymerase III requires the transcription initiation factor TFIIIB. TFIIIB has been shown to be a target of repression by tumor suppressors such as ARF, p53, RB, and the RB‐related pocket proteins. Also, TFIIIB activity is regulated by the oncogenes c‐Myc and the ERK mitogen‐activated protein kinase. PTEN is a commonly mutated tumor suppressor regulating cell growth, proliferation, and survival. Thus, we examined the potential for PTEN to regulate gene external RNA polymerase III transcription. Here, we report that PTEN inhibits both gene internal and external RNA polymerase III transcription in a variety of cervical, breast and prostate cancer cells. Deletion analysis suggests that the phosphatase domain is important in regulating gene internal RNA polymerase III transcription and the C2‐lipid binding domain in PTEN is important in regulating gene external RNA polymerase III transcription, providing a novel mechanism by which PTEN regulates basal transcription. Additionally, we show that PTEN repression of gene external RNA polymerase III transcription occurs through TFIIIB.
Histone Acetyltransferase p300 and Ets‐1 Synergistically Activate Guanylyl Cyclase/Natriuretic Peptide Receptor‐A Gene Expression
Authors: Kumar Prerna , Pandey Kailash N.
Published: June 2021

Cardiac hormone atrial natriuretic peptide (ANP) plays a pivotal role in maintaining blood pressure and cardiovascular homeostasis. Activation of guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) by ANP produces the second messenger cGMP. The objective of the present study was to elucidate the mechanism of Npr1 (coding for GC‐A/NPRA) gene transcription and expression. The studies were carried out in cultured mouse mesangial cells (MMC) which were transiently transfected and dual luciferase activity was assayed in the cell lysate. The results showed that a 90 bp fragment (‐46 to +55) in the Npr1 promoter contains transcriptional coactivator p300 binding site. Deletion or mutation of the p300 binding site decreased the promoter activity by 50%. Chromatin immunoprecipitation assay showed in vivo binding of p300 with Npr1 promoter. Overexpression of wild‐type adenovirus E1A significantly decreased the Npr1 promoter activity by 40%, whereas the mutant E1A had no discernible effect. Moreover, p300 showed functional synergism with Ets‐1 and increased Npr1 gene transcription by almost 16‐fold, NPRA mRNA levels by 6‐fold, and ANP‐stimulated intracellular accumulation of cGMP levels by 26‐fold. The results indicate that the Npr1 gene transcription and expression are critically controlled by synergistic interaction of p300 and Ets‐1. The present findings should yield important insights into the molecular mechanisms governing Npr1 gene regulation and expression, an important locus in the control of hypertension and cardiovascular homeostasis.
Detection and Quantification of Large Scale Deletions of Human Mitochondrial DNA by Real‐time PCR
Authors: Qian Edan , Xue Julie , Guo James M. , Gao Xueshan , Eaton John W , Qian Mingwei
Published: June 2021

Large deletions of mitochondrial DNA (mtDNA) occur in mitochondrial disease, oxidative stress and aging. Three approaches were developed to identify and quantify the deletions in nine cell lines and cheek cells collected from 18 human volunteers.1) mtDNA samples were screened by qPCR (SYBR Green) with over 150 pairs of primers with expected amplicon sizes >1kb. The qPCR conditions were set to amplify short sequences only (<400 bp). 2) Additional primers flanking the possible deletion sequences were designed to pinpoint the deletion sites. 3) The deletions were normalized with two short amplicons of the mtDNA (<250 bp) and compared with two known common deletions (CDs): 4977‐bp and 3895‐bp. It was found that: 1) CD 4977 and CD 3895 were common in all cell lines and in cheek cells. 2) The percentages of the two CDs varied widely, from 0.1 and 3.8 to 2.2 and 14.6% in cell lines and, from 0.1 to 7.8 and 1.6 to 20.4% in cheek cells, for CD 4977 and CD 3895, respectively. Due to large inter‐individual variations, the differences in age groups were not significant (P>0.05). 3) A few deletions (2 ‐8 kb) in cell lines and in cheek cells were demonstrated in gels. In conclusion, we identified and quantified several mtDNA deletions in human cell lines and cheek cells by qPCR. Our results demonstrate that qPCR with optimized conditions could be used as a sensitive, precise, and cost‐effective assay for high throughput screening of mtDNA deletions.
Immunofluorescence detection applications using DyLightTM Fluorescent Conjugates
Authors: Nlend Marie , Dworecki Boguslawa , Desai Surbhi , Hong Suk Jin , Shiflett Steve , Bell Peter A.
Published: June 2021

Immunofluorescence detection has become a powerful tool in life science and biomedical research. Fluorescence enables uses in multiplexed and homogenous assays in broad range of linear detection, increased sensitivities, greater selectivity, higher signal‐to‐noise relative to colorimetric methods, and safe handling and disposal of fluorescent reagents. Thermo Scientific offers a number of amine and Sulfhydryl Reactive DyLight fluorophores and protein conjugates across the visible and infrared regions of the spectrum. DyLight conjugates generate bright fluorescence intensity, photostability during long image capture times, good water solubility, and pH insensitivity. The spectral separation of our dyes facilitates the design of multiple labeling experiments in sophisticated cell biology and biomedically relevant applications. The excitation and emission spectra of the DyLight dyes are compatible with common laser and filter sets. We demonstrated here the use of Thermo Scientific DyLight conjugates with high reproducibility in multiplex labeling experiments, using fluorescence microscopy, confocal microscopy, and immunohistochemistry. Thermo Scientific DyLight conjugates are also ideal for quantitative evaluation of biological processes using techniques such as high content screening, RNAi, flow cytometry, fluorescent western blotting, and in vivo imaging.
Elucidating the Global Oxygen Regulatory Network in Yeast.
Authors: Henke Robert Michael , Lee Hee Chul , Shah Ajit , Majumdar Ranita , Zhang Li
Published: June 2021

Understanding the mechanisms by which cells sense and respond to oxygen is a fundamental biological problem. Mechanisms of oxygen sensing and regulation underlie many physiological and pathological processes in living organisms. Saccharomyces Cerevisiae provides an excellent model system for studying oxygen sensing and regulation in eukaryotes. We have employed genomic, computational, proteomic and biochemical approaches to elucidate the global molecular mechanisms underlying oxygen regulation in yeast. By using a machine learning algorithm called MEDUSA to analyze genome‐wide gene expression data, we identified a series of potential regulators that can control oxygen regulation of gene expression. These regulators include known oxygen regulators, such as Upc2, Hap1, and Mga2, and many new oxygen regulators. Furthermore, by using experimental approaches, we identified these hypoxia‐responsive genes, the deletion of which enables cells to grow better under hypoxic conditions, and those whose cellular localization is altered upon changes of oxygen level. Results from these experiments are combined to delineate the global oxygen regulatory network in yeast.