Making a First Impression: Exploring What Instructors Do and Say on the First Day of Introductory STEM Courses.

Student impressions formed during the first day of class can impact course satisfaction and performance. Despite its potential importance, little is known about how instructors format the first day of class. Here, we report on observations of the first day of class in 23 introductory science, technology, engineering, and math (STEM) courses. We first described how introductory STEM instructors structure their class time by characterizing topics covered on the first day through inductive coding of class videos. We found that all instructors discussed policies and basic information. However, a cluster analysis revealed two groups of instructors who differed primarily in their level of STEM content coverage. We then coded the videos with the noncontent Instructor Talk framework, which organizes the statements instructors make unrelated to disciplinary content into several categories and subcategories. Instructors generally focused on building the instructor-student relationship and establishing classroom culture. Qualitative analysis indicated that instructors varied in the specificity of their noncontent statements and may have sent mixed messages by making negatively phrased statements with seemingly positive intentions. These results uncovered variation in instructor actions on the first day of class and can help instructors more effectively plan this day by providing messages that set students up for success.

"What Will I Experience in My College STEM Courses?" An Investigation of Student Predictions about Instructional Practices in Introductory Courses.

The instructional practices used in introductory college courses often differ dramatically from those used in high school courses, and dissatisfaction with these practices is cited by students as a prominent reason for leaving science, technology, engineering, and mathematics (STEM) majors. To better characterize the transition to college course work, we investigated the extent to which incoming expectations of course activities differ based on student demographic characteristics, as well as how these expectations align with what students will experience. We surveyed more than 1500 undergraduate students in large introductory STEM courses at three research-intensive institutions during the first week of classes about their expectations regarding how class time would be spent in their courses. We found that first-generation and first-semester students predict less lecture than their peers and that class size had the largest effect on student predictions. We also collected classroom observation data from the courses and found that students generally underpredicted the amount of lecture observed in class. This misalignment between student predictions and experiences, especially for first-generation and first-semester college students and students enrolled in large- and medium-size classes, has implications for instructors and universities as they design curricula for introductory STEM courses with explicit retention goals.

Thermal acclimation and gene expression in rainbow smelt: Changes in the myotomal transcriptome in the cold.

Rainbow smelt, Osmerus mordax, have an impressive ability to acclimate to very cold water. Rainbow smelt exposed to cold (<5 °C) for an extended period of time have faster sustained swimming speeds and increased contraction kinetics in their myotomal muscle compared to warm acclimated fish. We used RNA Sequencing reactions (RNA-Seq) to explore how gene expression underlies thermal acclimation by muscle in these fish. Transcriptome analysis is limited in species that lack an annotated genome, such as rainbow smelt. The Trinity software package permits the de novo assembly of a rainbow smelt transcriptome with a modest learning curve. The transcriptome was then analyzed with Kallisto to quantify the abundance of each transcript represented in the full transcriptome and Sleuth to analyze the resulting RNA-seq datasets. Subsequently qPCR was used to explore patterns of thermal acclimation and gene expression for genes of metabolic and muscle contractile function. These methodologies revealed shifts in both muscle and metabolic gene expression that contribute to the thermal acclimation response in rainbow smelt. In fast-twitch, anaerobic white muscle, slow isoforms of myosin heavy and light chain tended to be down-regulated with exposure to cold in myotomal muscle, while fast isoforms were unchanged. Genes associated with protein turnover and aerobic metabolism were up-regulated in the white muscle, while those associated with anaerobic metabolism and the cell cycle were down-regulated. Collectively the results suggest that thermal acclimation to cold is complex process of apparent shifts in gene expression.

Functional interaction of bZIP proteins and the large subunit of replication factor C in liver and adipose cells.

The transcription factor CCAAT/enhancer-binding protein-alpha (C/EBPalpha) has a vital role in cell growth and differentiation. To delineate further a mechanism for C/EBPalpha-mediated differentiation, we screened C/EBPalpha-interacting proteins through far-Western screening. One of the strongest interactions was with RFC140, the large subunit of the replication factor C complex. C/EBPalpha specifically interacted with RFC140 from rat liver nuclear extract as determined by a combination of affinity chromatography and co-immunoprecipitation. Subsequent far-Western blotting showed that the bZIP domain of C/EBPalpha interacted with the DNA-binding region of RFC140. Overexpression of RFC140 in mammalian cells increased the transactivation activity of C/EBPalpha on both minimal and native promoters. Consistent with the enhanced transactivation, a complex of C/EBPalpha and RFC140 proteins with the cognate DNA element was detected in vitro. The specific interaction between C/EBPalpha and RFC140 was detected in the terminal differentiation of 3T3-L1 preadipocytes to adipocytes. The synergistic transcription effect of these two proteins increased the promoter activity and protein expression of peroxisome proliferator-activated receptor-gamma, which is a main regulator of adipocyte differentiation. Our results demonstrate that the specific transcription factor C/EBPalpha and the general DNA replication factor RFC140 interact functionally and physically. This observation highlights a unique mechanism by which the levels of the general replication factor can strongly modulate the functional activity of the specific transcription factor as a coactivator.

Who/se we are: baptism as personhood.

The attempt to arrive at some consensus on precisely what qualifies a human as a persons represents one of the more persistently debated and widely significant issues in modern biomedical ethics. The attribution of personhood has been and continues to be a powerful tool in moral discourse. Biomedical and bioethical debates about personhood seem especially morally significant in late modernity given the recent trends in biomedical technology. Our attempts to formally articulate universally agreed upon criteria for personhood represent some of the last vestiges of the hope that we can achieve substantial moral agreement in an otherwise morally fragmented world. In this essay, we argue that, from the perspective of certain strands of the Christian tradition, all bioethics grounded in attempts to develop formal, objective criteria by which we may designate a given individual a person are misguided. Criteria centering on the possession of reflective mental capacity, moreover, are for Christians especially problematic. We suggest that there are no morally neutral ways of designating personhood.

Human glioma-induced immunosuppression involves soluble factor(s) that alters monocyte cytokine profile and surface markers.

Patients with gliomas exhibit deficient in vitro and in vivo T cell immune activity, and human glioblastoma culture supernatants (GCS) inhibit in vitro T lymphocyte responses. Because APC are essential for initiating and regulating T cell responses, we investigated whether GCS would affect cytokines produced by monocytes and T cells from healthy donors of PBMC. Incubation of PBMC with GCS decreased production of IL-12, IFN-gamma, and TNF-alpha, and increased production of IL-6 and IL-10. The GCS-induced changes in IL-12 and IL-10 occurred in monocytes, and involved changes in IL-12 p40 and IL-10 mRNA expression. Incubation with GCS also resulted in reduced expression of MHC class II and of CD80/86 costimulatory molecules on monocytes. The immunosuppressive effects were not the result of IL-6 or TGF-beta1 that was detected in GCS. However, it was due to a factor(s) that is resistant to pH extremes, differentially susceptible to temperature, susceptible to trypsin, and has a minimum molecular mass of 40 kDa. Our findings show that glioblastoma-generated factors that are known to suppress T cell responses alter the cytokine profiles of monocytic APC that, in turn, inhibit T cell function. This model indicates that monocytes can serve as an intermediate between tumor-generated immune-suppressive factors and the T cell responses that are suppressed in gliomas.

Activating transcription factor-2 regulates phosphoenolpyruvate carboxykinase transcription through a stress-inducible mitogen-activated protein kinase pathway.

Several protein-nucleic acid complexes are observed when nuclear extracts from hepatoma cells are assayed for binding to the cAMP response element found in the phosphoenolpyruvate carboxykinase-cytosolic (PEPCK-C) promoter. Although cAMP response element-binding protein and CCAAT/enhancer binding proteins alpha and beta have been identified as liver factors that bind this motif, an uncharacterized, slower migrating complex was also observed. We identify activating transcription factor-2 (ATF-2) as the factor in this complex and show that ATF-2 stimulates expression from the PEPCK-C promoter. ATF-2 is a basic-leucine zipper transcription factor and a target for stress-activated protein kinases. We demonstrate that p38beta mitogen-activated protein (MAP) kinase augments ATF-2 transactivation activity on the PEPCK-C promoter, which is consistent with the interpretation that PEPCK-C promoter activity is maintained under stress through a p38 MAP kinase dependent pathway. In this regard, we show that treatment with sodium arsenite, a known activator of p38 MAP kinases, also stimulates expression from the PEPCK promoter. These results show that ATF-2 can stimulate transcription of the PEPCK-C promoter and support a role for stress inducible kinases in the maintenance of PEPCK-C expression.

ATF-2 and C/EBPalpha can form a heterodimeric DNA binding complex in vitro. Functional implications for transcriptional regulation.

We screened an expression cDNA library with a radiolabeled C/EBPalpha fusion protein and isolated three independent cDNAs encoding ATF-2, a bZIP protein that binds cAMP response elements (CRE). This interaction requires the respective bZIP domains, which form a typical bZIP heterodimer with altered DNA binding selectivity. C/EBPalpha and ATF-2 homodimers bind CRE sites, but ATF-2:C/EBPalpha heterodimers do not. Heterodimers bind an asymmetric sequence composed of one consensus half-site for each monomer, and may thus have a unique regulatory function. As predicted, co-transfection of ATF-2 with C/EBPalpha results in decreased activation of transcription driven from consensus C/EBP-binding sites. In contrast, C/EBPalpha and ATF-2 function cooperatively to activate transcription driven by the asymmetric sequence. Both factors are expressed in liver, where immunoprecipitation experiments show that ATF-2 co-precipitates with C/EBPalpha. These results are consistent with the interpretation that C/EBPalpha and ATF-2 can associate in vivo. Moreover, the formation of ATF-2:C/EBPbeta heterodimers suggests that cross-family dimerization with ATF-2 may be a general property for C/EBP family proteins.

L-arginine decreases infarct size in rats exposed to environmental tobacco smoke.

This study examined the effects of L-arginine on myocardial infarct size, hemodynamics, and vascular reactivity in environmental tobacco smoke (ETS)-exposed and non-ETS-exposed rats. We previously demonstrated that exposure to ETS increased myocardial infarct size in a rat model of ischemia and reperfusion. If reduced reperfusion was caused by endothelial cell damage and increased vascular tone, L-arginine (ARG) would increase nitric oxide and better protect the heart. Sixty Sprague-Dawley rats were randomly divided into four groups: ETS or non-ETS (control) with and without ARG (2.25% ARG in drinking water). The ETS groups were exposed to passive smoking (4 Marlboro cigarettes per 15 minutes, 6 hours a day) for 6 weeks. After 6 weeks, all rats were subjected to 35 minutes of left coronary artery occlusion and 120 minutes of reperfusion, with hemodynamic monitoring. Aortic rings were harvested to evaluate vascular reactivity. Average air nicotine, carbon monoxide, and total particulate concentrations were 1304 +/- 215 microgram/m3, 78 +/- 2.0 ppm, and 31 +/- .7 mg/m3 (mean +/- SEM) for the ETS-exposed rats. Infarct size (infarct mass/risk area x 100%) increased with ETS exposure but decreased significantly in the ETS-with-ARG group compared with the ETS-without-ARG group (42% +/- 6% vs 64% +/- 6%, mean +/- SEM; p = 0.043). The benefit of ARG was dependent on ETS exposure (ETS x ARG interaction, p = 0.043). There were no significant differences between groups in heart rate, systolic pressure, and rate-pressure product. ARG significantly decreased myocardial infarct size after ischemia and reperfusion in ETS-exposed rats. Neither the adverse effects of ETS on infarct size nor the blockage of this effect by ARG appears to be the result of ETS-induced alterations in hemodynamics.

Purification of a novel MHC class I element binding activity from thymus nuclear extracts reveals that thymic RBP-Jkappa/CBF1 binds to NF-kappaB-like elements.

We purified a DNA binding protein that recognizes a portion of the MHC class I regulatory element region 1/NF-kappaB binding site whose expression correlates with the expression of a MHC class I transgene in the thymus. The N-terminal amino acid sequence and the molecular size matched the RBP-Jkappa protein, also known as the EBV C-promoter binding factor, CBF1. Anti-peptide sera reactive with RBP-Jkappa/CBF1 also reacted with this protein in gel mobility shift assays. Although RBP-Jkappa/CBF1 is ubiquitously expressed, binding to the MHC class Ia NF-kappaB site was limited to the thymus. Comparison of the DNA binding specificities of RBP-Jkappa/CBF1 in thymic and splenic nuclear extracts revealed strong binding from both extracts to an IFN-beta kappaB site containing the RBP-Jkappa/CBF1 consensus sequence (CGTGGGAA). In contrast, only the thymic nuclear extract showed strong DNA binding activity with probes containing the NF-kappaB recognition sequences present in the MHC class Ia, IL-2Ralpha, and granulocyte-macrophage CSF promoters. Thus, RBP-Jkappa/CBF1 in thymic extracts demonstrates a clearly distinguishable DNA binding specificity that correlates with tissue-specific expression of a class I transgene. This, coupled with the fact that our previous study showed enhanced expression of the transgene in CD4+CD8+ thymocytes, suggests that RBP-Jkappa/CBF1 may play a role in the development of the immune system.

Phosphorylation of CCAAT-enhancer binding protein by protein kinase C attenuates site-selective DNA binding.

Four DNA-recombinant proteins, corresponding to the DNA-binding domain of CCAAT/enhancer binding protein (C/EBP), were phosphorylated in vitro by protein kinase C (PKC). High-performance liquid chromatography-peptide mapping of 32P-labeled C/EBP indicated the presence of three major 32P-labeled peptides: S299 (P)RDK, AKKS277 (P)VDK, and GAAGLPGPGGS248 (P)LK. Phosphorylation of C/EBP by PKC or M-kinase resulted in an attenuation of binding to a 32P-labeled CCAAT oligodeoxynucleotide. Three other truncated forms of C/EBP, C/EBP87, C/EBP87S-C, and C/EBP60, were studied to define the sites of phosphorylation affecting DNA binding. Phosphorylation of the C/EBP87, containing sites Ser299 and Ser277, and C/EBP60, containing only site Ser299, by PKC also resulted in attenuation of DNA binding. In contrast, phosphorylation of C/EBP87S-C, which retained Ser277 but had a Cys in place of Ser299, had no effect on DNA binding. Ser299 could not be phosphorylated by PKC if the protein is already bound to specific DNA. Phosphorylation of intact C/EBP from liver nuclear extract by PKC or M-kinase occurred at Ser299 and Ser277 and at an additional site, as demonstrated by immunoprecipitation and peptide mapping.

DNA-induced increase in the alpha-helical content of C/EBP and GCN4.

Leucine zipper proteins comprise a recently identified class of DNA binding proteins that contain a bipartite structural motif consisting of a "leucine zipper" dimerization domain and a segment rich in basic residues responsible for DNA interaction. Protein fragments encompassing the zipper plus basic region domains (bZip) have previously been used to determine the conformational and dynamic properties of this motif. In the absence of DNA, the coiled-coil portion is alpha-helical and dimeric, whereas the basic region is flexible and partially disordered. Addition of DNA containing a specific recognition sequence induces a fully helical conformation in the basic regions of these fragments. However, the question remained whether the same conformational change would be observed in native bZip proteins where the basic regions might be stabilized in an alpha-helical conformation even in the absence of DNA, through interactions with portions of the protein not included in the bZip motif. We have now examined the DNA-induced conformational transition for an intact bZip protein, GCN4, and for the bZip fragment of C/EBP with two enhancers that are differentially symmetric. Our results are consistent with the induced helical fork model wherein the basic regions are largely flexible in the absence of DNA and become fully helical in the presence of the specific DNA recognition sequence.

The role of the CCAAT/enhancer-binding protein in the transcriptional regulation of the gene for phosphoenolpyruvate carboxykinase (GTP).

Previous studies have identified a region in the promoter of the gene for phosphoenolpyruvate carboxykinase (GTP) (PEPCK) (positions -460 to +73) containing the regulatory elements which respond to cyclic AMP, glucocorticoids, and insulin and confer the tissue- and developmental stage-specific properties to the gene. We report that CCAAT/enhancer-binding protein (C/EBP) binds to the cyclic AMP-responsive element CRE-1 as well as to two regions which have been previously shown to bind proteins enriched in liver nuclei. The DNase I footprint pattern provided by the recombinant C/EBP was identical to that produced by a 43-kDa protein purified from rat liver nuclear extracts, using a CRE oligonucleotide affinity column, which was originally thought to be the CRE-binding protein CREB. Transient contransfection experiments using a C/EBP expression vector demonstrated that C/EBP could trans activate the PEPCK promoter. The trans activation occurred through both the upstream, liver-specific protein-binding domains and the CRE. The CRE-binding protein bound only to CRE-1 and not to the upstream C/EBP-binding sites. The results of this study, along with physiological properties of C/EBP, indicate an important role for this transcription factor in providing the PEPCK gene with several of its regulatory characteristics.

Evidence of changes in protease sensitivity and subunit exchange rate on DNA binding by C/EBP.

The transcription factor C/EBP uses a bipartite structural motif to bind DNA. Two protein chains dimerize through a set of amphipathic alpha helices termed the leucine zipper. Highly basic polypeptide regions emerge from the zipper to form a linked set of DNA contact surfaces. In the recently proposed a "scissors grip" model, the paired set of basic regions begin DNA contact at a central point and track in opposite directions along the major groove, forming a molecular clamp around DNA. This model predicts that C/EBP must undertake significant changes in protein conformation as it binds and releases DNA. The basic region of ligand-free C/EBP is highly sensitive to protease digestion. Pronounced resistance to proteolysis occurred when C/EBP associated with its specific DNA substrate. Sequencing of discrete proteolytic fragments showed that prominent sites for proteolysis occur at two junction points predicted by the "scissors grip" model. One junction corresponds to the cleft where the basic regions emerge from the leucine zipper. The other corresponds to a localized nonhelical segment that has been hypothesized to contain an N-cap and facilitate the sharp angulation necessary for the basic region to track continuously in the major groove of DNA.

Ig/EBP-1: a ubiquitously expressed immunoglobulin enhancer binding protein that is similar to C/EBP and heterodimerizes with C/EBP.

We report the isolation and characterization of cDNA clones that encode a protein with the same DNA binding specificity as the immunoglobulin heavy chain enhancer binding protein E (muEBP-E). We call the gene encoding this protein Ig/EBP-1. A fusion protein encoded by the cDNA binds specifically to muEBP-E-binding sites (E sites) in both the IgH enhancer and the VH1 promoter. Sequence analysis reveals that Ig/EBP-1 is a member of the "basic-zipper" family of DNA-binding proteins that are characterized by basic regions and heptad repeats of leucine residues. Among known family members, Ig/EBP-1 demonstrates highest homology to C/EBP throughout the DNA-binding domain and leucine repeat region. Ig/EBP-1 and C/EBP have highly overlapping binding specificities; both cloned proteins bind to the IgH enhancer and the VH1 promoter E sites, and Ig/EBP-1 binds to previously characterized C/EBP binding sites in the Rous sarcoma virus (RSV) LTR and the murine albumin promoter. Consistent with their homology in the leucine repeat region, Ig/EBP-1 and C/EBP form heterodimers; Ig/EBP-1 is the first member of this family that has been found to heterodimerize with the well-characterized C/EBP. Ig/EBP-1 mRNA is present in all tissues and cell lines examined, although its levels vary almost 20-fold from different sources, with highest levels in early B cells. In tissues where Ig/EBP-1 and C/EBP are both present, heterodimers may be functionally important. The presence of Ig/EBP-1 in fibroblasts and other tissues where C/EBP is not expressed suggests that Ig/EBP-1 may be functionally important for the activity of the RSV enhancer in these cell types. Finally, elevated expression of Ig/EBP-1 in early B cells may explain in part the enhancer-independent activity of VH promoters early in B-cell development.

Expression of the 31-kD stress protein in rat myoblasts and hepatocytes.

Rat tissue cultures cells respond to stress by inducing the synthesis of about 20 proteins, including two low-molecular-weight species of about 31 kD and 27 kD. We have cloned a cDNA for the 31-kD protein. This protein is induced in myoblasts and hepatoma cells in response to a 43 degrees C heat shock, or exposure to sodium arsenite or cadmium chloride salts. Furthermore, this protein is superinduced in hepatoma cells conditioned to grow in cadmium and zinc salts when they are exposed to a standard sodium arsenite stress. Induction of the gene encoding the 31-kD protein has been characterized as follows: (i) Transcripts accumulate maximally with similar kinetics when myoblasts are induced with either heat shock or sodium arsenite; (ii) accumulation of transcripts decays to preinduction levels within 4 hr of a heat shock, but requires more than 8 days after sodium arsenite stress; (iii) basal levels of transcript are reduced when myoblasts are cultured in the presence of steroid hormones; and (iv) stress induction is virtually abolished once myoblasts have differentiated.