Randomized Controlled Trial of a Cohesive Eight-Week Evolution Unit That Incorporates Molecular Genetics and Principles of the Next Generation Science Standards.

In response to calls for curricular materials that integrate molecular genetics and evolution and adhere to the K-12 Next Generation Science Standards (NGSS), the Genetic Science Learning Center (GSLC) at the University of Utah has developed and tested the "Evolution: DNA and the Unity of Life" curricular unit for high school biology. The free, 8-week unit illuminates the underlying role of molecular genetics in evolution while providing scaffolded opportunities to engage in making arguments from evidence and analyzing and interpreting data.  We used a randomized controlled trial design to compare student learning when using the new unit with a condition in which teachers used their typical (NGSS-friendly) units with no molecular genetics. Results from nationwide testing with 38 teachers (19 per condition) and their 2269 students revealed that students who used the GSLC curriculum had significantly greater pre/post gain scores in their understanding of evolution than students in the comparison condition; the effect size was moderate. Further, teacher implementation data suggest that students in the treatment condition had more opportunities to engage in argumentation from evidence and have in-class discussions than students in the comparison classes. We consider study implications for the secondary and postsecondary science education community.

Abnormal development of kitten retino-geniculate connectivity in the absence of action potentials.

Action potentials were silenced in one eye of neonatal kittens by repeated intraocular injections of tetrodotoxin for 5 to 8 weeks. After tetrodotoxin blockade was allowed to wear off, receptive field properties of individual relay cells in the lateral geniculate nucleus were examined. The many ON-OFF and binocular fields found in the layers that receive input from the treated eye suggest that these cells had extremely abnormal retino-geniculate synaptic connections. These effects were different in kind from those seen after deprivation rearing that does not silence action potentials. Lack of action potential activity was concluded to lead to abnormal development in the central nervous system.

Suppression of intestinal and mammary neoplasia by lifetime administration of aspirin in Apc(Min/+) and Apc(Min/+), Msh2(-/-) mice.

Numerous studies have indicated that exposure to nonsteroidal anti-inflammatory drugs is associated with a lowered risk of colorectal cancer. However, analyses of the effect of aspirin upon tumorigenesis in Apc(Min/+) mice have yielded contrasting results. We show that adult dietary exposure to aspirin does not suppress intestinal tumorigenesis in Apc(Min/+) mice, but that continual exposure from the point of conception does. To test whether this regime could suppress the phenotype of murine models of hereditary nonpolyposis colorectal cancer, Msh2-deficient mice were exposed to aspirin. This did not modify the mutator phenotype of Msh2(-/-) mice, but weakly extended survival. Finally, we analyzed (Apc(Min/+), Msh2(-/-)) mice and found that lifetime aspirin exposure significantly delayed the onset of both intestinal and mammary neoplasia. Thus embryonic and perinatal exposure to aspirin suppresses neoplasia specifically associated with the loss of Apc function, opening a potential window of opportunity for nonsteroidal anti-inflammatory drug intervention.

Response properties of neurons in the lateral geniculate nucleus of neonatal kittens.

A previous study led us to consider the implications of the types of receptive fields found in the lateral geniculate nucleus (LGN) of neonatal kittens. Thus, we studied cells in the A layers in the LGN of kittens aged 6-29 days using extracellular recording techniques. Peri-stimulus-time-histograms were constructed in response to flashing spots of light centered in the receptive field of each unit. All units studied showed an excitatory response only to light onset (on-center) or light offset (off-center). No units were found which had an excitatory response to both phases of the stimulus (On-Off). Possible differences in classification between this study and that of earlier workers who reported On-Off cells in young kittens are discussed.

Presence and distribution of Borrelia burgdorferi sensu lato species in internal organs and skin of naturally infected symptomatic and asymptomatic dogs, as detected by polymerase chain reaction.

Tissues from Dutch family dogs symptomatic for borreliosis according to established criteria and from infected but asymptomatic dogs were tested for Borrelia burgdorferi sensu lato DNA using a polymerase chain reaction. Subsequently, B. burgdorferi sensu stricto, B. garinii, B. afzelii, and B. valaisiana were identified by hybridization. Symptomatic dogs showed a higher prevalence of Borrelia in liver samples (9 of 15) than asymptomatic dogs (9 of 43) p = 0.0049. Overall, B. garinii was the most prevalent species and occurred together with up to three other species in on liver sample. B. burgdorferi sensu stricto however, was predominantly detected in samples of synovial membranes, skin, cerebrospinal fluid, bladder, heart, and bone marrow. Nine out of 10 symptomatic dogs with a very high antibody titre were positive for Borrelia DNA by PCR in one or more of these tissues. We conclude that dissemination in naturally infected European dogs occurs and that the two most prevalent species, B. burgdorferi sensu stricto and B. garinii, differ in their tropism.

Nucleolar NF-κB/RelA mediates apoptosis by causing cytoplasmic relocalization of nucleophosmin.

In a number of contexts, and particularly in response to cellular stress, stimulation of the NF-kappaB (NF-κB) pathway promotes apoptosis. One mechanism underlying this pro-apoptotic activity is nucleolar sequestration of RelA, which is reported to cause cell death by repressing NF-κB-driven transcription. Here, we identify a novel and distinct nucleolar activity of RelA that induces apoptosis. We demonstrate, using a viral nucleolar localization signal (NoLS)-RelA fusion protein, that direct targeting of RelA to the nucleolus mediates apoptosis, independent of NF-κB transcriptional activity. We demonstrate a requirement for nucleophosmin (NPM, B23.1) in this apoptotic effect, and the apoptotic effect of stress-induced nucleolar RelA. We show by multiple approaches that nucleolar translocation of RelA is causally involved in the relocalization of NPM from the nucleolus to the cytoplasm and that RelA-induced cytoplasmic NPM mediates apoptosis by facilitating the mitochondrial accumulation of BAX. These data uncover a novel stress-response pathway and mechanism by which RelA promotes apoptosis, independent of its effects on NF-κB transcriptional activity. These findings are relevant to the design of novel anticancer agents that target RelA to this compartment.

The proapoptotic effects of sulindac, sulindac sulfone and indomethacin are mediated by nucleolar translocation of the RelA(p65) subunit of NF-kappaB.

Understanding the mechanisms that underlie the antitumour activity of non-steroidal anti-inflammatory drugs (NSAIDs) against colorectal cancer will allow the development of more effective and specific chemopreventative agents. Modulation of the NF-kappaB pathway has been implicated as a key effector of the antitumour effect of aspirin, but the effects of non-aspirin NSAIDs on this pathway have yet to be fully defined. Here, we demonstrate that sulindac, sulindac sulfone and indomethacin activate the NF-kappaB pathway in colorectal cancer cells, as determined by western blot analysis of cytoplasmic levels of IkappaBalpha and immunocytochemical analysis of nuclear NF-kappaB/RelA. Furthermore, we show that all of these NSAIDs induce nucleolar translocation of the RelA subunit of NF-kappaB. Using RelA deleted for the previously described nucleolar localization signal, we demonstrate that this response is causally involved in the apoptotic effects of these agents. Finally, we demonstrate that NSAID-mediated nucleolar translocation of RelA is associated with downregulation of NF-kappaB-driven transcription and of the NF-kappaB target gene, ICAM-1. These data identify nucleolar translocation of RelA and the associated repression of the NF-kappaB-driven transcription as a central molecular mechanism of NSAID-mediated growth inhibition and apoptosis. As well as providing new understanding of the molecular determinants of RelA function, these findings also have relevance to the development of novel chemotherapeutic and chemopreventative agents.

Aspirin-induced nuclear translocation of NFkappaB and apoptosis in colorectal cancer is independent of p53 status and DNA mismatch repair proficiency.

Substantial evidence indicates nonsteroidal anti-inflammatory drugs (NSAIDs) protect against colorectal cancer (CRC). However, the molecular basis for this anti-tumour activity has not been fully elucidated. We previously reported that aspirin induces signal-specific IkappaBalpha degradation followed by NFkappaB nuclear translocation in CRC cells, and that this mechanism contributes substantially to aspirin-induced apoptosis. We have also reported the relative specificity of this aspirin-induced NFkappaB-dependent apoptotic effect for CRC cells, in comparison to other cancer cell types. It is now important to establish whether there is heterogeneity within CRC, with respect to the effects of aspirin on the NFkappaB pathway and apoptosis. p53 signalling and DNA mismatch repair (MMR) are known to be deranged in CRC and have been reported as potential molecular targets for the anti-tumour activity of NSAIDs. Furthermore, both p53 and MMR dysfunction have been shown to confer resistance to chemotherapeutic agents. Here, we set out to determine the p53 and hMLH1 dependency of the effects of aspirin on NFkappaB signalling and apoptosis in CRC. We specifically compared the effects of aspirin treatment on cell viability, apoptosis and NFkappaB signalling in an HCT-116 CRC cell line with the p53 gene homozygously disrupted (HCT-116(p53-/-)) and an HCT-116 cell line rendered MMR proficient by chromosomal transfer (HCT-116(+ch3)), to the parental HCT-116 CRC cell line. We found that aspirin treatment induced apoptosis following IkappaBalpha degradation, NFkappaB nuclear translocation and repression of NFkappaB-driven transcription, irrespective of p53 and DNA MMR status. These findings are relevant for design of both novel chemopreventative agents and chemoprevention trials in CRC.

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) interacts with Lys-tRNA synthetase: implications for priming of HIV-1 reverse transcription.

The vpr gene of human immunodeficiency virus type 1 (HIV-1) encodes a 96-amino-acid 14-kDa protein (viral protein R [Vpr]), which is produced late in the viral life cycle and is incorporated into the virion. Although Vpr is not required for viral replication in transformed cell lines and primary T lymphocytes, it is essential for productive infection of macrophages and monocytes and appears to be important for pathogenesis in vivo. To establish the role of Vpr in HIV-1 replication and pathogenesis, we have isolated cellular proteins with which Vpr interacts. By using the yeast two-hybrid system, Lys-tRNA synthetase (LysRS) was identified as a Vpr-interacting protein. The interaction between Vpr and LysRS was characterized both in vitro and in vivo, and the domains of Vpr required for the interaction were defined. In the presence of Vpr, LysRS-mediated amino-acylation of tRNA(Lys) is inhibited. Since tRNA(Lys) is the primer for reverse transcription of the HIV-1 genome, this suggests that the interaction between Vpr and LysRS may influence the initiation of HIV-1 reverse transcription.

A role for action-potential activity in the development of neuronal connections in the kitten retinogeniculate pathway.

The role of action potentials in the development of proper synaptic connections in the mammalian CNS was studied in the kitten retinogeniculate pathway. Our basic finding is that there is improper segregation of retinal inputs onto LGN cells after prolonged retinal action-potential blockade. Retinal ganglion cell firing was silenced from birth by repeated monocular injections of TTX. The resulting ganglion cell connections in the LGN were studied electrophysiologically after the action-potential blockade was ended. Most cells in the deprived LGN layers received excitatory input from both ON-center and OFF-center type ganglion cells, whereas LGN cells normally receive inputs only from ON-center or OFF-center ganglion cells, but not from both types. Improper segregation of ON and OFF inputs has never been reported after other types of visual deprivation that do not block ganglion cell activity. Control experiments showed that receptive fields in the nondeprived LGN layers were normal, that ganglion cell responses remained normal, and that there was no obvious ganglion cell loss. We also showed that individual LGN cells with ON and OFF excitatory inputs were not present in normal neonatal kittens. Two other types of improper input segregation in response to action-potential blockade were also found in the deprived LGN layers. (1) A greater than normal number of LGN cells received both X- and Y-type ganglion cell input. (2) Almost half of the cells at LGN layer borders were excited binocularly. Recovery of LGN normality was rapid and complete after blockade that lasted for only 3 weeks from birth, but little recovery was seen after about 11 weeks of blockade. The susceptibility to action-potential blockade decreased during the first 3 postnatal weeks. These findings may result from axon-terminal sprouting or from the failure of axon terminals to retract. The results are consistent with the idea that normally synchronous activity of neighboring ganglion cells of like center-type may be used in the refinement of retinogeniculate synaptic connections.

Evidence for colorectal cancer cell specificity of aspirin effects on NF kappa B signalling and apoptosis.

Epidemiological evidence indicates that non-steroidal anti-inflammatory drugs (NSAIDs) protect against colorectal cancer (CRC) to a greater degree than other non-gastrointestinal cancers, but the molecular basis for this difference is unknown. We previously reported that aspirin induces signal-specific I kappa B alpha degradation followed by NF kappa B nuclear translocation in CRC cells, and that this mechanism contributes substantially to aspirin-induced apoptosis. Here, we explored the hypothesis that cell-type specific effects on NF kappa B signalling are responsible for the observed differences in protection by aspirin against CRC compared to breast and gynaecological cancers. We also assessed whether COX-2 expression, mutation status of adenomatous polyposis coli (APC), beta-catenin, p53, or DNA mismatch repair (MMR) genes in CRC lines influenced aspirin-induced effects. We found that aspirin induced concentration-dependent I kappa B alpha degradation, NF kappa B nuclear translocation and apoptosis in all CRC lines studied. However, there was no such effect on the other cancer cell types, indicating a considerable degree of cell-type specificity. The lack of effect on NF kappa B signalling, paralleled by absence of an apoptotic response to aspirin in non-CRC lines, strongly suggests a molecular rationale for the particular protective effect of NSAIDs against CRC. Effects on NF kappa B and apoptosis were observed irrespective of COX-2 expression, or mutation status in APC, beta-catenin, p53 and DNA MMR genes, underscoring the generality of the aspirin effect on NF kappa B in CRC cells. These findings raise the possibility of cell-type specific targets for the development of novel chemopreventive agents.

Elimination of action potentials blocks the structural development of retinogeniculate synapses.

Although the influence of electrical activity on neural development has been studied extensively, experiments have only recently focused on the role of activity in the development of the mammalian central nervous system (CNS). Using tetrodotoxin (TTX) to abolish sodium-mediated action potentials, studies on the visual system show that impulse activity is essential both for the normal development of neuronal size and responsivity in the lateral geniculate nucleus (LGN), and for the eye-specific segregation of geniculo-cortical axons. There have been no anatomical studies to investigate the influence of action potentials on CNS synaptic development. We report here the first direct evidence that elimination of action potentials in the mammalian CNS blocks the growth of developing axon terminals and the formation of normal adult synaptic patterns. Our results show that when TTX is used to eliminate retinal ganglion-cell action potentials in the cat from birth to 8 weeks, the connections made by ganglion cell axons with LGN neurones, retinogeniculate synapses, remain almost identical morphologically to those in the newborn kitten.

Renal allograft recipients with high susceptibility to cutaneous malignancy have an increased prevalence of human papillomavirus DNA in skin tumours and a greater risk of anogenital malignancy.

Renal allograft recipients (RARs) have a well-documented increased incidence of viral warts and cutaneous neoplasia, particularly those with long graft life and high sun exposure. A clinicopathological survey of 69 RARs in south-east Scotland, with follow-up periods of up to 28 years after transplantation, revealed marked variation in patient susceptibility to cutaneous malignancy with concomitant variation in HPV prevalence. Skin cancers were found in 34 patients. Eight patients showed high susceptibility [defined as more than four intraepidermal carcinomas (IECs) or invasive squamous cell carcinomas (SCCs)] 42 had intermediate susceptibility (1-3 IECs or SCCs, or >3 keratoses) and 18 had low susceptibility (< or = 3 keratoses and no cancers). SCCs, IECs and keratoses from the high-susceptibility group were found to have greater prevalences of human papillomavirus (HPV) DNA (56%, 45% and 50% respectively), than SCCs (0%) and IECs (33%) from intermediate-susceptibility RARs and keratoses (36%) from the combined intermediate- and low-susceptibility groups and compared with a group of immunocompetent controls (27%, 20% and 15% respectively). No differences in p53 protein accumulation, determined immunohistochemically, were observed in tumours from the three groups. Categorization of RARs by susceptibility to cutaneous malignancy provides clinically useful information, as significantly more high-susceptibility patients (38%) developed aggressive, potentially lethal anogenital or cutaneous squamous cell cancers than did patients in the intermediate group (5%, P=0.005) or the low-susceptibility group (0%).

Prevalence of human papillomavirus DNA in cutaneous neoplasms from renal allograft recipients supports a possible viral role in tumour promotion.

It is well established that renal allograft recipients (RARs) have an increased incidence of viral warts and premalignant and malignant cutaneous lesions, and the risk of their development increases in proportion to duration of graft survival. It has been postulated that, in addition to the effects of prolonged immunosuppression and previous sun exposure, human papillomaviruses (HPV) may also contribute to the carcinogenic process. In this study, the prevalence of HPV DNA was examined in a range of premalignant and malignant cutaneous tumours from 50 immunosuppressed patients (47 renal allograft recipients plus three cardiac allograft recipients) and 56 immunocompetent patients using Southern hybridisation as a low-stringency screening method and type-specific polymerase chain reaction (PCR) assays for eight HPV types. The combined results for renal allograft recipients show that HPV DNA was detectable in 79% of viral warts, 42% of premalignant keratoses, 33% of intraepidermal carcinomas, 43% of invasive squamous cell carcinomas and 16% of uninvolved skin specimens (squamous cell carcinomas/renal allograft recipients significantly different at P < 0.05 from uninvolved skin specimens/renal allograft recipients). In immunocompetent patients the pattern of HPV DNA prevalence was 100% for viral warts; 25% for keratoses, 23% for intraepidermal carcinomas, 22% for squamous cell carcinomas and 8% for uninvolved skin. No single HPV type predominated in tumour specimens from either group. More tumours were found to contain HPV DNA by Southern hybridisation analysis than PCR, indicating the presence of HPV types other than HPV 1, 2, 5, 6, 8, 11, 16 and 18 in some tumours. However, 'low cancer risk' HPV types 1, 2 and 6 as well as 'high cancer risk' HPV types 5 and 16 were specifically detected by PCR in a small number of neoplasms. These data suggest that multiple HPV types may contribute to cutaneous neoplasia in RARs and that they appear to act early in the process of carcinogenesis, perhaps by functioning as tumour promoters via stimulation of cell proliferation.

Accumulation of p53 is associated with tumour progression in cutaneous lesions of renal allograft recipients.

Renal allograft recipients suffer from a markedly increased susceptibility to premalignant and malignant cutaneous lesions. Although various aetiological factors have been implicated, little is known of the associated genetic events. In this study we initially employed immunocytochemical techniques to investigate the prevalence and localisation of accumulated p53 in over 200 cutaneous biopsies (including 56 squamous cell carcinomas) from renal allograft recipients and immunocompetent controls. In renal allograft recipients accumulated p53 was present in 24% of uninvolved skin samples, 14% of viral warts, 41% of premalignant keratoses, 65% of intraepidermal carcinomas and 56% of squamous cell carcinomas [squamous cell carcinoma and intraepidermal carcinoma differed significantly from uninvolved skin (P < 0.005) and viral warts (P < 0.01)]. A similar trend was revealed in immunocompetent patients (an older, chronically sun-exposed population) but with lower prevalence of p53 immunoreactivity: 25% of uninvolved skin samples, 0% of viral warts, 25% of keratoses, 53% of intraepidermal carcinomas and 53% of squamous cell carcinomas. These differences were not statistically significant. Morphologically, p53 immunoreactivity strongly associated with areas of epidermal dysplasia and the abundance of staining correlated positively with the severity of dysplasia. These data suggest that p53 plays a role in skin carcinogenesis and is associated with progression towards the invasive state. No correlation was observed between accumulated p53 and the presence of human papillomavirus (HPV) DNA in any of the lesions. Single-strand conformational polymorphism analysis (exons 5-8) was used to determine the frequency of mutated p53 in 28 malignancies with varying degrees of immunopositivity. p53 mutations were found in 5/9 (56%) malignancies with p53 staining in > 50% of cells, reducing to 1/6 (17%) where 10-50% of cells were positively stained and none where < 10% of cells were stained. These data imply that factors other than p53 gene mutation play a part in accumulation of p53 in skin cancers.