High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

The University of Texas Medical School at Houston

Voicu and Schmajuk (Rob. Auto. Syst. 35 (2001a) 23) described a model of spatial navigation and exploration that includes an action system capable of guiding, with the help of a cognitive system, the search for specific goals as determined by a motivation system. Whereas in the original model the cognitive map stores information about the connectivity between places in the environment, in the present version the cognitive map also stores information about the paths traversed by the agent. Computer simulations show that the network correctly describes experimental results including latent learning in a maze, detours in a maze, and shortcuts in an open field. In addition, the model generates novel predictions about detours and shortcuts in an open field.

Latent learning, shortcuts and detours: a computational model.

We describe a computational model of spatial navigation based on experimental studies conducted with human participants. The model builds and uses a hierarchical cognitive map of a large environment. Computer simulations show that the model correctly describes experimental results including hierarchical organization of space and distance estimation. Furthermore, the model predicts that reaction time for distance estimation varies nonlinearly with distance.

Hierarchical cognitive maps.

Hippocampal neurons show a strong modulation by theta frequency oscillations. This modulation is thought to be important not only for temporal encoding and decoding of information in the hippocampal system, but also for temporal ordering of neuronal activities on timescales at which physiological mechanisms of synaptic plasticity operate. The medial entorhinal cortex (MEC), one of the two major cortical inputs to the hippocampus, is known to show theta modulation. Here, we show that the local field potentials (LFPs) in the other major cortical input to the hippocampus, the lateral entorhinal cortex (LEC), show weaker theta oscillations than those shown in the MEC. Neurons in LEC also show weaker theta modulation than that of neurons in MEC. These findings suggest that LEC inputs are integrated into hippocampal representations in a qualitatively different manner than the MEC inputs. Furthermore, MEC grid cells increase the scale of their periodic spatial firing patterns along the dorsoventral axis, corresponding to the increasing size of place fields along the septotemporal axis of the hippocampus. We show here a corresponding gradient in the tendency of MEC neural firing to skip alternate theta cycles. We propose a simple model based on interference of delta oscillations with theta oscillations to explain this behavior.

Theta modulation in the medial and the lateral entorhinal cortices.

Limited availability of in vitro and in vivo model systems has hampered efforts to understand tumor biology and test novel therapies for ependymoma, the third most common malignant brain tumor that occurs in children. To develop clinically relevant animal models of ependymoma, we directly injected a fresh surgical specimen from a 9-year-old patient into the right cerebrum of RAG2/severe complex immune deficiency (SCID) mice. All five mice receiving the initial transplantation of the patient tumor developed intracerebral xenografts, which have since been serially subtransplanted in vivo in mouse brains for 4 generations and can be cryopreserved for long-term maintenance of tumorigenicity. The xenograft tumors shared nearly identical histopathological features with the original tumors, harbored 8 structural chromosomal abnormalities as detected with spectral karyotyping, maintained gene expression profiles resembling that of the original patient tumor with the preservation of multiple key genetic abnormalities commonly found in human ependymomas, and contained a small population (<2.2%) of CD133(+) stem cells that can form neurospheres and display multipotent capabilities in vitro. The permanent cell line (BXD-1425EPN), which was derived from a passage II xenograft tumor and has been passaged in vitro more than 70 times, expressed similar differentiation markers of the xenograft tumors, maintained identical chromosomal abnormalities, and formed tumors in the brains of SCID mice. In conclusion, direct injection of primary ependymoma tumor cells played an important role in the generation of a clinically relevant mouse model IC-1425EPN and a novel cell line, BXD-1425EPN. This cell line and model will facilitate the biological studies and preclinical drug screenings for pediatric ependymomas.

A clinically relevant orthotopic xenograft model of ependymoma that maintains the genomic signature of the primary tumor and preserves cancer stem cells in vivo.

Long-term synaptic plasticity is believed to underlie the capacity for learning and memory. In the cerebellum, for example, long-term plasticity contributes to eyelid conditioning and to learning in eye movement systems. We report evidence for a decrementing form of cerebellar plasticity as revealed by the behavioral properties of eyelid conditioning in the rabbit. We find that conditioned eyelid responses exhibit within-session changes that recover by the next day. These changes, which increase with the interstimulus interval, involve decreases in conditioned response magnitude and likelihood as well as increases in latency to onset. Within-subject comparisons show that these changes differ in magnitude depending on the type of training, arguing against motor fatigue or changes in motor pathways downstream of the cerebellum. These phenomena are also observed when stimulation of mossy fibers substitutes for the conditioned stimulus, suggesting that changes take place within the cerebellum or in downstream efferent pathways. Together, these observations suggest a plasticity mechanism in the cerebellum that is induced during training sessions and fades within 23 h. To formalize this hypothesis more specifically, we show that incorporating a short-lasting potentiation at the granule cell to Purkinje cell synapses in a computer simulation of the cerebellum reproduces these behavioral effects. We propose the working hypothesis that the presynaptic form of long-term potentiation observed at these synapses is reversed by time rather than by a corresponding long-term depression. These results demonstrate the utility of eyelid conditioning as a means to identify and characterize the rules that govern input to output transformations in the cerebellum.

A decrementing form of plasticity apparent in cerebellar learning.

The temporally specific learning displayed by the cerebellum facilitates mechanistic analysis of neural timing and temporal coding. We report evidence for a subtraction-like mechanism of temporal coding in cerebellar cortex in which activity in a subset of granule cells specifically codes the interval between the offset of two mossy fiber inputs. In a large-scale cerebellar simulation, cessation of one of two ongoing mossy fiber inputs produces a robust temporal code in the population of granule cells. This activity supports simulation learning in response to temporal patterns of stimuli, even when those same stimuli do not support learning when presented individually. Using stimulation of mossy fiber inputs to the cerebellum as training stimuli in rabbits, we confirmed these unusual predictions in a cerebellum-dependent form of learning. Analysis of the simulations reveals a specific working hypothesis for this temporal subtraction process that involves interactions between granule cells and the inhibitory Golgi cells. The results suggest how feedforward inhibition, such as that present in the cerebellar cortex, can contribute to temporal coding.

A subtraction mechanism of temporal coding in cerebellar cortex.

We previously showed that primary tumor-based orthotopic xenograft mouse models of medulloblastoma replicated the histopathological phenotypes of patients' original tumors. Here, we performed global gene expression profiling of 11 patient-specific xenograft models to further determine whether the xenograft tumors were molecularly accurate during serial subtransplantations in mouse brains and whether they represented all the molecular subtypes of medulloblastoma that were recently described. Analysis of the transcriptomes of 9 pairs of matched passage I xenografts and patients' tumors revealed high correlation coefficients (r(2) > 0.95 in 5 models, > 0.9 in 3 models, and > 0.85 in 1 model) and only identified 69 genes in which expressions were altered (FDR = 0.0023). Subsequent pair-wise comparisons between passage I, III, and V xenografts from the 11 models further showed that no dramatic alterations were introduced (r(2) > 0.9 in 8 models and > 0.8 in 3 models). The genetic abnormalities of each model were then identified through comparison with control RNAs from 5 normal cerebella and 2 fetal brains. Hierarchical clustering using 3 previously published molecular signatures showed that our models span the whole spectrum of molecular subtypes, including SHH (n = 2), WNT (n = 2), and the most recently identified group C (n = 4) and group D (n = 3). In conclusion, we demonstrated that the 11 orthotopic medulloblastoma xenograft models were molecularly faithful to the primary tumors, and our comprehensive collection of molecularly distinct animal models should serve as a valuable resource for the development of new targeted therapies for medulloblastoma.

Global gene expression profiling confirms the molecular fidelity of primary tumor-based orthotopic xenograft mouse models of medulloblastoma.

Aberrant expression and activation of oncogenes in somatic cells has been associated with cancer initiation. Required for reacquisition of pluripotency in the developing germ cell, PRDM14 initiates lymphoblastic leukemia when misexpressed in murine bone marrow. Activation of pluripotency in somatic cells can lead to aneuploidy and copy number alterations during iPS cell generation, and we hypothesized that PRDM14-induced lymphoblastic leukemias would demonstrate significant chromosomal damage. High-resolution oligo array comparative genomic hybridization demonstrated infrequent aneuploidy but frequent amplification and deletion, with amplifications occurring in a 5:1 ratio with deletions. Many deletions (i.e., Cdkn2a, Ebf1, Pax5, Ikzf1) involved B-cell development genes and tumor suppressor genes, recapitulating deletions occurring in human leukemia. Pathways opposing senescence were frequently deactivated via Cdkn2a deletion or Tbx2 amplification, with corollary gene expression. Additionally, gene expression studies of abnormal pre-leukemic B-precursors showed downregulation of genes involved in chromosomal stability (i.e., Xrcc6) and failure to upregulate DNA repair pathways. We propose a model of leukemogenesis, triggered by pluripotency genes like Prdm14, which involves ongoing DNA damage and failure to activate non-homologous end-joining secondary to aberrant gene expression.

Mouse Lymphoblastic Leukemias Induced by Aberrant Prdm14 Expression Demonstrate Widespread Copy Number Alterations Also Found in Human ALL.

The cells present in amniotic fluid (AF) are currently used for prenatal diagnosis of fetal anomalies but are also a potential source of cells for cell therapy. To better characterize putative progenitor cell populations present in AF, we used culture conditions that support self-renewal to determine if these promoted the generation of stable cell lines from AF-derived cells (AFC). Cells isolated from E11.5 mouse were cultured on irradiated STO fibroblast feeder layers in human embryonic germ cell derivation conditions. The cultures grew multicellular epithelial colonies that could be repropagated from single cells. Reverse transcription semiquantitative polymerase chain reaction of established cell lines revealed that they belonged to the extraembryonic endoderm (ExEn) expressing high levels of Gata6, Gata4, Sox17, Foxa2 and Sox7 mRNA. Hierarchical clustering based on the whole transcriptome expression profile of the AFC lines (AFCL) shows significant correlation between transcription profiles of AFCL and blastocyst-derived XEN, an ExEn cell line. In vitro differentiation of AFCL results in the generation of cells expressing albumin and α-fetoprotein (AFP), while intramuscular injection of AFCL into immunodeficient mice produced AFP-positive tumors with primitive endodermal appearance. Hence, E11.5 mouse AF contains cells that efficiently produce XEN lines. These AF-derived XEN lines do not spontaneously differentiate into embryonic-type cells but are phenotypically stable and have the capacity for extensive expansion. The lack of requirement for reprogramming factors to turn AF-derived progenitor cells into stable cell lines capable of massive expansion together with the known ability of ExEn to contribute to embryonic tissue suggests that this cell type may be a candidate for banking for cell therapies.

Culture of mouse amniotic fluid-derived cells on irradiated STO feeders results in the generation of primitive endoderm cell lines capable of self-renewal in vitro.

Clinical outcome of children with malignant glioma remains dismal. Here, we examined the role of over-expressed BMI1, a regulator of stem cell self-renewal, in sustaining tumor formation in pediatric glioma stem cells. Our investigation revealed BMI1 over-expression in 29 of 54 (53.7%) pediatric gliomas, 8 of 8 (100%) patient derived orthotopic xenograft (PDOX) mouse models, and in both CD133+ and CD133- glioma cells. We demonstrated that lentiviral-shRNA mediated silencing of suppressed cell proliferation in vitro in cells derived from 3 independent PDOX models and eliminated tumor-forming capacity of CD133+ and CD133- cells derived from 2 PDOX models in mouse brains. Gene expression profiling showed that most of the molecular targets of BMI1 ablation in CD133+ cells were different from that in CD133- cells. Importantly, we found that silencing BMI1 in CD133+ cells derived from 3 PDOX models did not affect most of the known genes previously associated with the activated BMI1, but modulated a novel set of core genes, including RPS6KA2, ALDH3A2, FMFB, DTL, API5, EIF4G2, KIF5c, LOC650152, C20ORF121, LOC203547, LOC653308, and LOC642489, to mediate the elimination of tumor formation. In summary, we identified the over-expressed BMI1 as a promising therapeutic target for glioma stem cells, and suggest that the signaling pathways associated with activated BMI1 in promoting tumor growth may be different from those induced by silencing BMI1 in blocking tumor formation. These findings highlighted the importance of careful re-analysis of the affected genes following the inhibition of abnormally activated oncogenic pathways to identify determinants that can potentially predict therapeutic efficacy.

Silencing BMI1 eliminates tumor formation of pediatric glioma CD133+ cells not by affecting known targets but by down-regulating a novel set of core genes.

Rhabdoid histology in clear-cell renal cell carcinoma is associated with a poor prognosis. The prognosis of patients with clear-cell renal cell carcinoma may also be influenced by molecular alterations. The aim of this study was to evaluate the association between histologic features and salient molecular changes in rhabdoid clear-cell renal cell carcinoma. We macrodissected the rhabdoid and clear-cell epithelioid components from 12 cases of rhabdoid clear-cell renal cell carcinoma. We assessed cancer-related mutations from eight cases using a clinical next-generation exome-sequencing platform. The transcriptome of rhabdoid clear-cell renal cell carcinoma (n=8) and non-rhabdoid clear-cell renal cell carcinoma (n=37) was assessed by RNA-seq and gene expression microarray. VHL (63%) showed identical mutations in all regions from the same tumor. BAP1 (38%) and PBRM1 (13%) mutations were identified in the rhabdoid but not in the epithelioid component and were mutually exclusive in 3/3 cases and 1 case, respectively. SETD2 (63%) mutations were discordant between different histologic regions in 2/5 cases, with mutations called only in the epithelioid and rhabdoid components, respectively. The transcriptome of rhabdoid clear-cell renal cell carcinoma was distinct from advanced-stage and high-grade clear-cell renal cell carcinoma. The diverse histologic components of rhabdoid clear-cell renal cell carcinoma, however, showed a similar transcriptomic program, including a similar prognostic gene expression signature. Rhabdoid clear-cell renal cell carcinoma is transcriptomically distinct and shows a high rate of SETD2 and BAP1 mutations and a low rate of PBRM1 mutations. Driver mutations in clear-cell renal cell carcinoma are often discordant across different morphologic regions, whereas the gene expression program is relatively stable. Molecular profiling of clear-cell renal cell carcinoma may improve by assessing for gene expression and sampling tumor foci from different histologic regions.

Intratumoral morphologic and molecular heterogeneity of rhabdoid renal cell carcinoma: challenges for personalized therapy.

Sarcomatoid transformation, wherein an epithelioid carcinomatous tumour component coexists with a sarcomatoid histology, is a predictor of poor prognosis in clear cell renal cell carcinoma. Our understanding of sarcomatoid change has been hindered by the lack of molecular examination. Thus, we sought to characterize molecularly the biphasic epithelioid and sarcomatoid components of sarcomatoid clear cell renal cell carcinoma and compare them to non-sarcomatoid clear cell renal cell carcinoma. We examined the transcriptome of the epithelioid and sarcomatoid components of advanced stage sarcomatoid clear cell renal cell carcinoma (n=43) and non-sarcomatoid clear cell renal cell carcinoma (n=37) from independent discovery and validation cohorts using the cDNA microarray and RNA-seq platforms. We analyzed DNA copy number profiles, generated using SNP arrays, from patients with sarcomatoid clear cell renal cell carcinoma (n=10) and advanced non-sarcomatoid clear cell renal cell carcinoma (n=155). The epithelioid and sarcomatoid components of sarcomatoid clear cell renal cell carcinoma had similar gene expression and DNA copy number signatures that were, however, distinct from those of high-grade, high-stage non-sarcomatoid clear cell renal cell carcinoma. Prognostic clear cell renal cell carcinoma gene expression profiles were shared by the biphasic components of sarcomatoid clear cell renal cell carcinoma and the sarcomatoid component showed a partial epithelial-to-mesenchymal transition signature. Our genome-scale microarray-based transcript data were validated in an independent set of sarcomatoid and non-sarcomatoid clear cell renal cell carcinomas using RNA-seq. Sarcomatoid clear cell renal cell carcinoma is molecularly distinct from non-sarcomatoid clear cell renal cell carcinoma, with its genetic programming largely shared by its biphasic morphological components. These data explain why a low percentage of sarcomatoid histology augurs a poor prognosis; suggest the need to modify the pathological grading system and introduce the potential for candidate biomarkers to detect sarcomatoid change preoperatively without specifically sampling the histological sarcomatoid component.

Biphasic components of sarcomatoid clear cell renal cell carcinomas are molecularly similar to each other, but distinct from, non-sarcomatoid renal carcinomas.

Horatiu Voicu

Department of Biochemistry and Molecular Biology

The University of Texas Medical School at Houston

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

Horatiu Voicu

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biochemistry and Molecular Biology

The University of Texas Medical School at Houston

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

Department of Biochemistry and Molecular Biology