John Kratz

Comparative genomic hybridization detects an increased number of chromosomal alterations in large cell/anaplastic medulloblastomas.

We correlate chromosomal changes in medulloblastomas with histologic subtype, reporting the analysis of 33 medulloblastoma specimens by comparative genomic hybridization, and a subset by fluorescence in situ hybridization. Of the 33 tumors, 5 were desmoplastic/nodular, 10 were histologically classic, and 18 were large cell/anaplastic. Chromosomal gains and losses were more common in anaplastic medulloblastomas than in non‐anaplastic ones. We identified 4 medulloblastomas with c‐myc amplification and 5 medulloblastomas with N‐myc amplification; all 9 were of the large cell/anaplastic subtype. Additional regions with high level gains included 2q14‐22, 3p23, 5p14‐pter, 8q24, 9p22‐23, 10p12‐pter, 12q24, 12p11‐12, 17p11‐12, and Xp11. The majority of these high level gains occurred in anaplastic cases. We also found loss of chromosome 17p in 7 large cell/anaplastic cases but no non‐anaplastic medulloblastomas. Finally, we detected a significantly increased overall number of chromosomal alterations in large cell/anaplastic medulloblastomas (6.8/case) compared to non‐anaplastic ones (3.3/case). These findings support an association between myc oncogene amplification, 17p loss, and large cell/anaplastic histology.

hTERT gene amplification and increased mRNA expression in central nervous system embryonal tumors

High-level gains at 5p15, a chromosomal region including the human telomerase catalytic protein subunit (hTERT) gene, have been documented in several medulloblastomas. We therefore analyzed hTERT gene dosage in a group of medulloblastomas and other embryonal brain tumors using differential PCR. Amplification of the hTERT locus was detected in 15 of 36 (42%) tumors examined. To correlate gene amplification with message level, we used real-time quantitative PCR to measure hTERT mRNA in 50 embryonal brain tumors. hTERT mRNA was detected in all but one of these cases, and mRNA level correlated significantly with gene dosage (r = 0.82). Log-rank analysis of survival data revealed a trend toward poor clinical outcomes in patients with medulloblastomas containing high hTERT mRNA levels, but clinical follow-up was relatively short and the association was not statistically significant (P = 0.078). Comparative genomic hybridization was used to further analyze the tumor with the greatest hTERT gene dosage and mRNA level, a recurrent medulloepithelioma. hTERT was amplified in the recurrent tumor but not in the primary lesion, suggesting this locus can be involved in tumor progression. Our data indicate that hTERT gene amplification is relatively common in embryonal brain tumors, and that increased expression of hTERT mRNA may be associated with biologically aggressive tumor behavior.

Microtubule depolymerization in Caenorhabditis elegans touch receptor neurons reduces gene expression through a p38 MAPK pathway

Microtubules are integral to neuronal development and function. They endow cells with polarity, shape, and structure, and their extensive surface area provides substrates for intracellular trafficking and scaffolds for signaling molecules. Consequently, microtubule polymerization dynamics affect not only structural features of the cell but also the subcellular localization of proteins that can trigger intracellular signaling events. In the nematode Caenorhabditis elegans, the processes of touch receptor neurons are filled with a bundle of specialized large-diameter microtubules. We find that conditions that disrupt these microtubules (loss of either the MEC-7 β-tubulin or MEC-12 α-tubulin or growth in 1 mM colchicine) cause a general reduction in touch receptor neuron (TRN) protein levels. This reduction requires a p38 MAPK pathway (DLK-1, MKK-4, and PMK-3) and the transcription factor CEBP-1. Cells may use this feedback pathway that couples microtubule state and MAPK activation to regulate cellular functions.

Data publication consensus and controversies

The movement to bring datasets into the scholarly record as first class research products (validated, preserved, cited, and credited) has been inching forward for some time, but now the pace is quickening. As data publication venues proliferate, significant debate continues over formats, processes, and terminology. Here, we present an overview of data publication initiatives underway and the current conversation, highlighting points of consensus and issues still in contention. Data publication implementations differ in a variety of factors, including the kind of documentation, the location of the documentation relative to the data, and how the data is validated. Publishers may present data as supplemental material to a journal article, with a descriptive “data paper,” or independently. Complicating the situation, different initiatives and communities use the same terms to refer to distinct but overlapping concepts. For instance, the term published means that the data is publicly available and citable to virtually everyone, but it may or may not imply that the data has been peer-reviewed. In turn, what is meant by data peer review is far from defined; standards and processes encompass the full range employed in reviewing the literature, plus some novel variations. Basic data citation is a point of consensus, but the general agreement on the core elements of a dataset citation frays if the data is dynamic or part of a larger set. Even as data publication is being defined, some are looking past publication to other metaphors, notably “data as software,” for solutions to the more stubborn problems.

Researcher perspectives on publication and peer review of data

Data “publication” seeks to appropriate the prestige of authorship in the peer-reviewed literature to reward researchers who create useful and well-documented datasets. The scholarly communication community has embraced data publication as an incentive to document and share data. But, numerous new and ongoing experiments in implementation have not yet resolved what a data publication should be, when data should be peer-reviewed, or how data peer review should work. While researchers have been surveyed extensively regarding data management and sharing, their perceptions and expectations of data publication are largely unknown. To bring this important yet neglected perspective into the conversation, we surveyed ∼ 250 researchers across the sciences and social sciences– asking what expectations“data publication” raises and what features would be useful to evaluate the trustworthiness, evaluate the impact, and enhance the prestige of a data publication. We found that researcher expectations of data publication center on availability, generally through an open database or repository. Few respondents expected published data to be peer-reviewed, but peer-reviewed data enjoyed much greater trust and prestige. The importance of adequate metadata was acknowledged, in that almost all respondents expected data peer review to include evaluation of the data’s documentation. Formal citation in the reference list was affirmed by most respondents as the proper way to credit dataset creators. Citation count was viewed as the most useful measure of impact, but download count was seen as nearly as valuable. These results offer practical guidance for data publishers seeking to meet researcher expectations and enhance the value of published data.

A Disease-causing Mutation Illuminates the Protein Membrane Topology of the Kidney-expressed Prohibitin Homology (PHB) Domain Protein Podocin

Background: Mutations in the stomatin family protein podocin are the most common genetic cause of proteinuria. Results: A conserved proline residue of podocin is essential for its membrane topology. Conclusion: This study confirms a hairpin-like structure of the membrane-attached PHB domain protein and its significance for cholesterol recruitment. Significance: PodocinP118L elucidates the pathogenic implication in kidney disease and identifies a novel family of PHB domain proteins. Mutations in the NPHS2 gene are a major cause of steroid-resistant nephrotic syndrome, a severe human kidney disorder. The NPHS2 gene product podocin is a key component of the slit diaphragm cell junction at the kidney filtration barrier and part of a multiprotein-lipid supercomplex. A similar complex with the podocin ortholog MEC-2 is required for touch sensation in Caenorhabditis elegans. Although podocin and MEC-2 are membrane-associated proteins with a predicted hairpin-like structure and amino and carboxyl termini facing the cytoplasm, this membrane topology has not been convincingly confirmed. One particular mutation that causes kidney disease in humans (podocinP118L) has also been identified in C. elegans in genetic screens for touch insensitivity (MEC-2P134S). Here we show that both mutant proteins, in contrast to the wild-type variants, are N-glycosylated because of the fact that the mutant C termini project extracellularly. PodocinP118L and MEC-2P134S did not fractionate in detergent-resistant membrane domains. Moreover, mutant podocin failed to activate the ion channel TRPC6, which is part of the multiprotein-lipid supercomplex, indicative of the fact that cholesterol recruitment to the ion channels, an intrinsic function of both proteins, requires C termini facing the cytoplasmic leaflet of the plasma membrane. Taken together, this study demonstrates that the carboxyl terminus of podocin/MEC-2 has to be placed at the inner leaflet of the plasma membrane to mediate cholesterol binding and contribute to ion channel activity, a prerequisite for mechanosensation and the integrity of the kidney filtration barrier.

Making data count

Science is built on a foundation of data. The production and publication of that data should be recognized as valuable scholarship, but data lacks an essential prerequisite for modern-day scholarly recognition—accepted metrics of significance. The scientific community has traditionally estimated the impact of a journal article by counting the number of subsequent references to it; more recently, a suite of web-based alternative metrics (‘altmetrics’) offer faster assessment and the chance capture other kinds of impact1. Data can be fit into these article-centered assessment systems by proxy, via data descriptor articles in journals like Earth Systems Science Data or Scientific Data2,3. Another approach is to apply familiar metrics directly to datasets published in online databases or repositories. Complicating matters, the same metric may mean different things with respect to articles versus datasets. A researcher can read an article online closely without downloading the PDF version, but if they view a dataset landing page without downloading the data, their level of engagement is almost certainly low. A better understanding of how to measure data impact is critical if we are to reward data creators and incentivize data publication.

Prohibitin Homology Domain Proteins in Caenorhabditis elegans

PROHIBITIN HOMOLOGY DOMAIN PROTEINS IN CAENORHABDITIS ELEGANS John Ernest Kratz III The PHB-d protein family is an evolutionarily ancient family of integral membrane proteins with members in all taxa that are involved in a wide variety of biological process but share several common molecular properties: oligomerization, detergent resistant membrane (DRM) association, and regulation of other proteins. To better understand the biological roles of the PHB-d gene family and provide a starting point for analysis of their individual functions, I determined the expression patterns of all of the uncharacterized PHB-d genes in the nematode C. elegans. All eight of the proteins similar to mammalian stomatin are detectably expressed in neurons. The specificity of expression varies from 34 neuron types that express sto-4 to only one that expresses sto3. STO-1 protein is localized to amphid sensory cilia and is needed for optimal chemotaxis to diacetyl. This phenotype is likely to affect chemotaxis mediated by the AWA sensory neurons. Involvement of PHB-d proteins in chemosensation may be conserved in mammals. Two murine PHB-d genes are highly expressed in olfactory sensory neurons, but no olfactory phenotype has been described in mice lacking either gene. C. elegans senses gentle body touch via a mechanosensory channel complex that contains two Degenerin/Epithelial sodium channel (DEG/ENaC) subunits and two prohibitin homology domain (PHB-d) proteins. One of the PHB-d proteins (UNC-24) has a C-terminal sterol carrier protein 2 (SCP-2) domain and only a subtle mechanosensory abnormal (Mec) phenotype. The other PHB-d (MEC-2) is essential for channel function. I show here that unc-24 is needed for proper localization of MEC-2 to the channel complex, but that this function is unlikely to underlie the unc-24 phenotype. This result supports the hypothesis that PHB-d/SCP-2 proteins have a conserved role in localizing other PHB-d proteins and demonstrates that UNC-24 must have a second, unknown, role in mechanosensation. MEC-2 is embedded in the plasma membrane via a non-spanning hydrophobic hook. Mutation of a conserved proline (P134S) in the hydrophobic hook leads to complete touch insensitivity. Mutation of the equivalent proline in human stomatin converts the hook to a transmembrane domain and expels the PHB-d and C-terminus of the protein. We show here that the P134S mutation has the same effect on MEC-2 topology in HEK293T cells. This result has implications for MEC-2 function because the P134S mutation has previously been shown to affect some but not all MEC-2 biochemical properties. The conserved proline is found in all C. elegans and human stomatins and its mutation in human podocin leads to kidney failure.