Transfer-bound community college students' biology identity and perception of teaching.

Community colleges are frequently an affordable, accessible entrance to a Science, Technology, Engineering, and Mathematics (STEM) education and career, but the transition from a 2-year program to a 4-year institution can be tumultuous. In this mixed-methods study, we explore the experiences of transfer and prospective transfer students. Through surveys and interviews, we identify the challenges faced by and the supports desired by biology transfer students. We describe how community college students perceive their introductory biology courses, and we compare the biology identity and self-efficacy of these students to peers at a 4-year institution. Students expressed uncertainty about what to expect from the transfer experience, and they benefitted from interventions that made the university experience more concrete or clarified their expectations. We found that community college students are just as interested in biology as peers at a 4-year university, but they are significantly less likely to believe that others recognize them as "biology people" and report less self-efficacy regarding biology courses. Students felt particularly well-prepared for transfer after community college biology courses they described as "rigorous" and "demanding," especially because students expressed that the community college environment helped support them through the challenges of higher education.

Quantitative analysis and comparison of 3D morphology between viable and apoptotic MCF-7 breast cancer cells and characterization of nuclear fragmentation.

Morphological changes in apoptotic cells provide essential markers for defining and detection of apoptosis as a fundamental mechanism of cell death. Among these changes, the nuclear fragmentation and condensation have been regarded as the important markers but quantitative characterization of these changes is yet to be achieved. We have acquired confocal image stacks of 206 viable and apoptotic MCF-7 cells stained by three fluorescent dyes. Three-dimensional (3D) parameters were extracted to quantify and compare their differences in morphology. To analyze nuclear fragmentation, a new method has been developed to determine clustering of nuclear voxels in the reconstructed cells due to fluorescence intensity changes in nuclei of apoptotic cells. The results of these studies reveal that the 3D morphological changes in cytoplasm and nuclear membranes in apoptotic cells provide sensitive targets for label-free detection and staging of apoptosis. Furthermore, the clustering analysis and morphological data on nuclear fragmentation are highly useful for derivation of optical cell models and simulation of diffraction images to investigate light scattering by early apoptotic cells, which can lead to future development of label-free and rapid methods of apoptosis assay based on cell morphology.

Effect of Ca(2+) on Aß40 fibrillation is characteristically different.

Alzheimer's disease (AD) is the only one among top ten diseases in USA that cannot be cured, prevented or slowed down. At molecular level the mechanism of onset has been closely associated with mis-folding of Aß40 and Aß42 and is well supported by the genetic data for AD. Extensive research efforts have led to identification of factors and metal ions that could manipulate Aß equilibrium, especially Ca(2+). Previously, we reported selectively acceleration of Aß42 fibril formation by Ca(2+)in vitro within physiological concentrations (BBA (2009) 1794:1536). Aß40 on the other hand did not appear to be significantly affected by Ca(2+) addition. In an effort to understand the distinctive behavior of Aß40, we monitored changes of Aß40 aggregation by intrinsic tyrosine fluorescence and CD and took different approaches for data processing. Our analysis of CD data indicates a complex effect induced by the addition of 2mM Ca(2+) resulting in an increase in the rate of transformation from monomer to ß-sheet rich fibrilar or intermediate species formation in Aß40. Surprisingly, the kinetics observed by intrinsic fluorescence studies in this article and ThT, SEC or EM studies in our previous report were not able to unravel the existence of this effect in Aß40.

Spatio-temporal expression patterns of anterior Hox genes during Nile tilapia (Oreochromis niloticus) embryonic development.

Hox genes encode transcription factors that function to pattern regional tissue identities along the anterior-posterior axis during animal embryonic development. Divergent nested Hox gene expression patterns within the posterior pharyngeal arches may play an important role in patterning morphological variation in the pharyngeal jaw apparatus (PJA) between evolutionarily divergent teleost fishes. Recent gene expression studies have shown the expression patterns from all Hox paralog group (PG) 2-6 genes in the posterior pharyngeal arches (PAs) for the Japanese medaka (Oryzias latipes) and from most genes of these PGs for the Nile tilapia (Oreochromis niloticus). While several orthologous Hox genes exhibit divergent spatial and temporal expression patterns between these two teleost species in the posterior PAs, several tilapia Hox gene expression patterns from PG3-6 must be documented for a full comparative study. Here we present the spatio-temporal expression patterns of hoxb3b, c3a, b4a, a5a, b5a, b5b, b6a and b6b in the neural tube and posterior PAs of the Nile tilapia. We show that several of these tilapia Hox genes exhibit divergent expression patterns in the posterior PAs from their medaka orthologs. We also compare these gene expression patterns to orthologs in other gnathostome vertebrates, including the dogfish shark.

Role of Hox PG2 genes in Nile tilapia pharyngeal arch specification: implications for gnathostome pharyngeal arch evolution.

Phylogenetic reconstructions suggest that the ancestral osteichthyan Hox paralog group 2 gene complement was composed of two genes, Hoxa2 and b2, both of which have been retained in tetrapods, but only one of which functions as a selector gene of second pharyngeal arch identity (PA2). Genome duplication at the inception of the teleosts likely generated four Hox PG2 genes, only two of which, hoxa2b and b2a, have been preserved in zebrafish, where they serve as functionally redundant PA2 selector genes. Evidence from our laboratory has shown that other telelosts, specifically striped bass and Nile tilapia, harbor three transcribed Hox PG2 genes, hoxa2a, a2b, and b2a, with unspecified function(s). We have focused on characterizing the function of the three Nile tilapia Hox PG2 genes as a model to examine the effects of postgenome duplication gene loss on the evolution of developmental gene function. We studied Hox PG2 gene function in tilapia by examining the effects of independent morpholino oligonucleotide (MO)-induced knockdowns on pharyngeal arch morphology and Hox gene expression patterns. Morphological defects resulting from independent MO-induced knockdowns of tilapia hoxa2a, a2b, and b2a included the expected PA2 to PA1 homeotic transformations previously observed in tetrapods and zebrafish, as well as concordant and unexpected morphological changes in posterior arch-derived cartilages. Of particular interest, was the observation of a MO-induced supernumerary arch between PA6 and PA7, which occurred concomitantly with other MO-induced pharyngeal arch defects. Beyond these previously unreported morphant-induced transformations, a comparison of Hox PG2 gene expression patterns in tilapia Hox PG2 morphants were indicative of arch-specific auto- and cross-regulatory activities as well as a Hox paralog group 2 interdependent regulatory network for control of pharyngeal arch specification.

Embryonic development and skeletogenesis of the pharyngeal jaw apparatus in the cichlid Nile tilapia (Oreochromis niloticus).

The evolution of a specialized pharyngeal jaw apparatus (PJA) has been argued to be the key evolutionary innovation that allowed the explosive adaptive radiation of cichlid fishes in East African lakes. Subsequent studies together with recent molecular phylogenies have shown that similar innovations evolved independently several times within the teleosts, which poses the questions: (1) how similar are the developmental mechanisms responsible for these changes in divergent taxa and (2) how did such complex features arise independently in evolution? A detailed knowledge of PJA development in cichlids and other teleosts is needed to address these questions. Here, we provide a detailed account of the development of the PJA in one species of cichlid, the Nile tilapia (Oreochromis niloticus), from the early segmentation and patterning of its embryonic precursors - pharyngeal arches 3 to 7 - to its ossification. We find that pharyngeal segmentation occurs sequentially from anterior to posterior during early segmentation stages through the mid-pharyngula period. We show a clear combinatorial code of Hox gene expression such that each posterior arch is defined by a distinctive Hox signature. Posterior arch chondrogenesis in tilapia is essentially complete by the end of the hatching period, and most elements become ossified over the next two days. Our results reveal that both the fusion of lower jaw bones and articulation between the neurocranium and upper jaws occur during post-embryonic development.

Compromised repair of clustered DNA damage in the human acute lymphoblastic leukemia MSH2-deficient NALM-6 cells.

Ionizing radiation (IR) induces two classes of complex DNA damage, double-strand breaks (DSBs) and non-DSB bi-stranded oxidative clustered DNA lesions (OCDLs). OCDLs may consist of single strand breaks (SSBs), oxidized purines/pyrimidines and abasic sites within 5-10bp. These significant biological lesions are hypothesized to challenge the repair machinery and carry a high mutagenic potential. MSH2, a classical DNA mismatch repair protein, has been also implicated in other repair pathways associated with DSB and base lesion processing. MSH2 mutations have been identified in acute lymphoblastic leukemia (ALL) patients as well as in other types of cancers. Our research model involves two precursors B (pre-B) ALL human cell lines, NALM-6 cells, homozygous null for MSH2, and wild type 697 cells. Using a modified version of neutral and alkaline single cell gel electrophoresis (SCGE) with Escherichia coli repair enzymes as damage probes, the processing capacity of single strand breaks (SSBs), DSBs and OCDLs was assessed in NALM-6 and 697 cells exposed to a radiotherapy relevant gamma-ray dose of 5Gy. Using reverse transcriptase PCR and Western blotting we verified the complete lack of expression of MSH2 in the NALM-6 cells at the transcriptional and translational level. No differences were measured between NALM-6 and 697 cells in the induction levels of SSBs, DSBs and OCDLs after exposure to gamma-rays. However, 697 cells repaired each lesion more efficiently with significant differences observed after 1-3h post-irradiation. Lastly, our results indicate a significantly higher population of apoptotic 697 cells compared to NALM-6 cells 6-24h post-irradiation. Our studies suggest that MSH2 is probably involved in the processing of the biologically significant clustered DNA damages as well as the execution of apoptosis induced by ionizing radiation.

Japanese medaka Hox paralog group 2: insights into the evolution of Hox PG2 gene composition and expression in the Osteichthyes.

Hox paralog group 2 (PG2) genes function to specify the development of the hindbrain and pharyngeal arch-derived structures in the Osteichthyes. In this article, we describe the cDNA cloning and embryonic expression analysis of Japanese medaka (Oryzias latipes) Hox PG2 genes. We show that there are only two functional canonical Hox genes, hoxa2a and b2a, and that a previously identified hoxa2b gene is a transcribed pseudogene, psihoxa2b. The functional genes, hoxa2a and b2a, were expressed in developing rhombomeres and pharyngeal arches in a manner that was relatively well conserved compared with zebrafish (Danio rerio) but differed significantly from orthologous striped bass (Morone saxatilis) and Nile tilapia (Oreochromis niloticus) genes, which, we suggest, may be owing to effects of post-genome duplication loss of a Hox PG2 gene in the medaka and zebrafish lineages. psihoxa2b was expressed at readily detectable levels in several noncanonical Hox expression domains, including the ventral aspect of the neural tube, the pectoral fin buds and caudal-most region of the embryonic trunk, indicative that regulatory control elements needed for spatio-temporal expression have diverged from their ancestral counterparts. Comparative expression analyses showed medaka hoxa2a and b2a expression in the 2nd pharyngeal arch (PA2) beyond the onset of chondrogenesis, which, according to previous hypotheses, suggests these genes function redundantly as selector genes of PA2 identity. We conclude that Hox PG2 gene composition and expression have diverged significantly during osteichthyan evolution and that this divergence in teleosts may be related to lineage-dependent differential gene loss following an actinopterygian-specific whole genome duplication.

Consequences of hoxb1 duplication in teleost fish.

Vertebrate evolution is characterized by gene and genome duplication events. There is strong evidence that a whole-genome duplication occurred in the lineage leading to the teleost fishes. We have focused on the teleost hoxb1 duplicate genes as a paradigm to investigate the consequences of gene duplication. Previous analysis of the duplicated zebrafish hoxb1 genes suggested they have subfunctionalized. The combined expression pattern of the two zebrafish hoxb1 genes recapitulates the expression pattern of the single Hoxb1 gene of tetrapods, possibly due to degenerative changes in complementary cis-regulatory elements of the duplicates. Here we have tested the hypothesis that all teleost duplicates had a similar fate post duplication, by examining hoxb1 genes in medaka and striped bass. Consistent with this theory, we found that the ancestral Hoxb1 expression pattern is subdivided between duplicate genes in a largely similar fashion in zebrafish, medaka, and striped bass. Further, our analysis of hoxb1 genes reveals that sequence changes in cis-regulatory regions may underlie subfunctionalization in all teleosts, although the specific changes vary between species. It was previously shown that zebrafish hoxb1 duplicates have also evolved different functional capacities. We used misexpression to compare the functions of hoxb1 duplicates from zebrafish, medaka and striped bass. Unexpectedly, we found that some biochemical properties, which were paralog specific in zebrafish, are conserved in both duplicates of other species. This work suggests that the fate of duplicate genes varies across the teleost group.

Comparative analysis of Hox paralog group 2 gene expression during Nile tilapia (Oreochromis niloticus) embryonic development.

The hindbrain and pharyngeal arch-derived structures of vertebrates are determined, at least in part, by Hox paralog group 2 genes. In sarcopterygians, the Hoxa2 gene alone appears to specify structures derived from the second pharyngeal arch (PA2), while in zebrafish (Danio rerio), either of the two Hox PG2 genes, hoxa2b or hoxb2a, can specify PA2-derived structures. We previously reported three Hox PG2 genes in striped bass (Morone saxatilis), including hoxa2a, hoxa2b, and hoxb2a and observed that only HoxA cluster genes are expressed in PA2, indicative that they function alone or together to specify PA2. In this paper, we present the cloning and expression analysis of Nile tilapia (Oreochromis niloticus) Hox PG2 genes and show that all three genes are expressed in the hindbrain and in PA2. The expression of hoxb2a in PA2 was unexpected given the close phylogenetic relationship of Nile tilapia and striped bass, both of which are members of the order Perciformes. A reanalysis of striped bass hoxb2a expression demonstrated that it is expressed in PA2 with nearly the same temporal and spatial expression pattern as its Nile tilapia ortholog. Further, we determined that Nile tilapia and striped bass hoxa2a orthologs are expressed in PA2 well beyond the onset of chondrogenesis whereas neither hoxa2b nor hoxb2a expression persist until this stage, which, according to previous hypotheses, suggests that hoxa2a orthologs in these two species function alone as selector genes of PA2 identity.

Differential expression of hoxa2a and hoxa2b genes during striped bass embryonic development.

Here, we report the cloning and expression analysis of two previously uncharacterized paralogs group 2 Hox genes, striped bass hoxa2a and hoxa2b, and the developmental regulatory gene egr2. We demonstrate that both Hox genes are expressed in the rhombomeres of the developing hindbrain and the pharyngeal arches albeit with different spatio-temporal distributions relative to one another. While both hoxa2a and hoxa2b share the r1/r2 anterior boundary of expression characteristic of the hoxa2 paralog genes of other species, hoxa2a gene expression extends throughout the hindbrain, whereas hoxa2b gene expression is restricted to the r2-r5 region. Egr2, which is used in this study as an early developmental marker of rhombomeres 3 and 5, is expressed in two distinct bands with a location and spacing typical for these two rhombomeres in other species. Within the pharyngeal arches, hoxa2a is expressed at higher levels in the second pharyngeal arch, while hoxa2b is more strongly expressed in the posterior arches. Further, hoxa2b expression within the arches becomes undetectable at 60hpf, while hoxa2a expression is maintained at least up until the beginning of chondrogenesis. Comparison of the striped bass HoxA cluster paralog group 2 (PG2) genes to their orthologs and trans-orthologs shows that the striped bass hoxa2a gene expression pattern is similar to the overall expression pattern described for the hoxa2 genes in the lobe-finned fish lineage and for the hoxa2b gene from zebrafish. It is notable that the pharyngeal arch expression pattern of the striped bass hoxa2a gene is more divergent from its sister paralog, hoxa2b, than from the zebrafish hoxa2b gene. Overall, our results suggest that differences in the Hox PG2 gene complement of striped bass and zebrafish affects both their rhombomeric and pharyngeal arch expression patterns and may account for the similarities in pharyngeal arch expression between striped bass hoxa2a and zebrafish hoxa2b.

Cloning of somatolactin alpha and beta cDNAs in zebrafish and phylogenetic analysis of two distinct somatolactin subtypes in fish.

Somatolactin (SL) is a pituitary hormone belonging to the growth hormone/prolactin superfamily, with recognizable homologues in all fish taxa examined to date. Although sequences from most fish share reasonably high sequence identity, several more highly divergent SLs have been reported. Goldfish SL and a second SL protein found in rainbow trout (rtSLP) are remarkably different from each other and also dissimilar to other SLs. It has been unclear whether rtSLP is a recent paralogue restricted to rainbow trout, or reflects a more ancient duplication of the SL gene, and whether it is related to the goldfish sequence. Here we report the cloning of two different zebrafish SL cDNAs, which share only 57.5% nucleotide and 47.7% deduced amino acid identities. One copy, designated zebrafish SLalpha (zfSLalpha), displays a typical range of sequence similarity to most other SLs. The other copy, zebrafish SLbeta (zfSLbeta), shows low identity to most other SLs; surprisingly, it is most similar to the divergent SL sequence from goldfish. The mRNAs of zfSLalpha and zfSLbeta were expressed specifically in two distinct regions of the pars intermedia in zebrafish. Cells expressing zfSLalpha are located at the posterior pars intermedia, bordering the neurohypophysis, whereas zfSLbeta is expressed in the anterior part of the pars intermedia, bordering the pars distalis. Phylogenetic analyses indicate that zfSLbeta, goldfish SL and rtSLP all belong to the SL hormone family; however, along with the genes from eel and catfish, these divergent sequences form a group that is clearly distinct from all other SLs. These results suggest the presence of two distinct SL families, SLalpha and SLbeta, which may trace back to a teleost genome duplication prior to divergence of the cyprinids and salmonids.

Bichir HoxA cluster sequence reveals surprising trends in ray-finned fish genomic evolution.

The study of Hox clusters and genes provides insights into the evolution of genomic regulation of development. Derived ray-finned fishes (Actinopterygii, Teleostei) such as zebrafish and pufferfish possess duplicated Hox clusters that have undergone considerable sequence evolution. Whether these changes are associated with the duplication(s) that produced extra Hox clusters is unresolved because comparison with basal lineages is unavailable. We sequenced and analyzed the HoxA cluster of the bichir (Polypterus senegalus), a phylogenetically basal actinopterygian. Independent lines of evidence indicate that bichir has one HoxA cluster that is mosaic in its patterns of noncoding sequence conservation and gene retention relative to the HoxA clusters of human and shark, and the HoxAalpha and HoxAbeta clusters of zebrafish, pufferfish, and striped bass. HoxA cluster noncoding sequences conserved between bichir and euteleosts indicate that novel cis-sequences were acquired in the stem actinopterygians and maintained after cluster duplication. Hence, in the earliest actinopterygians, evolution of the single HoxA cluster was already more dynamic than in human and shark. This tendency peaked among teleosts after HoxA cluster duplication.

Evolutionary divergence of vertebrate Hoxb2 expression patterns and transcriptional regulatory loci.

Hox gene expression is regulated by a complex array of cis-acting elements that control spatial and temporal gene expression in developing embryos. Here, we report the isolation of the striped bass Hoxb2a gene, comparison of its expression to the orthologous gene from zebrafish, and comparative genomic analysis of the upstream regulatory region to that of other vertebrates. Comparison of the Hoxb2a gene expression patterns from striped bass to zebrafish revealed similar expression patterns within rhombomeres 3, 4, and 5 of the hindbrain but a notable absence of expression in neural crest tissues of striped bass while neural crest expression is observed in zebrafish and common to other vertebrates. Comparative genomic analysis of the striped bass Hoxb2a-b3a intergenic region to those from zebrafish, pufferfish, human, and mouse demonstrated the presence of common Meis, Hox/Pbx, Krox-20, and Box 1 elements, which are necessary for rhombomere 3, 4, and 5 expression. Despite their common occurrence, the location and orientation of these transcription elements differed among the five species analyzed, such that Krox-20 and Box 1 elements are located 3' to the Meis, Hox/Pbx elements in striped bass, pufferfish, and human while they are located 5' of this r4 enhancer in zebrafish and mouse. Our results suggest that the plasticity exhibited in the organization of key regulatory elements responsible for rhombomere-specific Hoxb2a expression may reflect the effects of stabilizing selection in the evolution cis-acting elements.

Heterogeneity of chondroitin sulfate glycosaminoglycan localization during early development of the striped bass (Morone saxatilis).

Recent studies have suggested important functions for proteoglycan-associated chondroitin sulfate glycosaminoglycans (GAGs) during embryonic and larval development in numerous organisms, including the teleost. Little is known, however, about the specific distribution of different chondroitin sulfate GAGs during early development. The present study utilized immunohistochemistry to localize chondroitin sulfate GAG antigens during development of the striped bass (Morone saxatilis). Immunoreagents utilized were monoclonal antibodies (MAbs) TC2, d1C4, and CS-56, which recognize, respectively, native epitopes on glycosaminoglycan chains enriched in chondroitin-4-, chondroitin-6-, and both chondroitin-4- and -6-sulfate. Little or no immunoreactivity was observed in gastrulating embryos at 18 hr postfertilization with any MAb tested. By 24 hr (8 somites), the CS-56 epitope was localized around the notochord. At hatching (48 hr) and early larval (72 hr) stages, d1C4 and CS-56 antigens codistributed in some sites (e.g., the notochord and myosepta), but a striking heterogeneity of chondroitin sulfate GAG localization was observed in other developing tissues, including the eye and specific subsets of basement membrane. At these latter time points, TC2 reacted primarily with the extracellular matrix of the developing heart, particularly the ventricular and conotruncal segments. Heterogeneous patterning of these chondroitin sulfate GAG epitopes suggests dynamic regulation of proteoglycan function during critical morphogenetic events in early development of the striped bass.