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The mitochondrial ribosome (mitoribosome) has diverged drastically from its evolutionary progenitor, the bacterial ribosome. Structural and compositional diversity is particularly striking in the phylum Euglenozoa, with an extraordinary protein gain in the mitoribosome of kinetoplastid protists. Here we report an even more complex mitoribosome in diplonemids, the sister-group of kinetoplastids. Affinity pulldown of mitoribosomal complexes from Diplonema papillatum, the diplonemid type species, demonstrates that they have a mass of > 5 MDa, contain as many as 130 integral proteins, and exhibit a protein-to-RNA ratio of 11:1. This unusual composition reflects unprecedented structural reduction of ribosomal RNAs, increased size of canonical mitoribosomal proteins, and accretion of three dozen lineage-specific components. In addition, we identified >50 candidate assembly factors, around half of which contribute to early mitoribosome maturation steps. Because little is known about early assembly stages even in model organisms, our investigation of the diplonemid mitoribosome illuminates this process. Together, our results provide a foundation for understanding how runaway evolutionary divergence shapes both biogenesis and function of a complex molecular machine.

Introduction Glioblastoma (GBM) is the most aggressive primary brain cancer. Identifying novel markers which stratify patients and predict response to targeted therapies is a priority. Telomeres are caps on chromosome ends protecting them from aberrant DNA damage response. Telomere length (TL) alterations and fusions are key markers for genomic instability and high-resolution telomere analysis provides powerful prognostic and predictive information in several tumour types. We therefore studied telomeric profiles and fusions in GBM patients and related this to clinical outcome. Methods Tumours from 151 adults with isocitrate dehydrogenase-wildtype GBMs were analysed for TL using the precision High-Throughput Single Telomere Length Analysis technique. 67 patients underwent further multiplex long-range polymerase chain reaction to identify fusions via Southern hybridisation using telomere-adjacent 33P radioisotope-labelled probes. Telomere fusions from 10 tumours that exhibited the highest frequency of fusions were characterised with long-read Nanopore sequencing. Results GBMs display short telomeres with a median length of 4.0kb. Shorter TLs in patients with unmethylated O6-methylguanine DNA methyltransferase status (commonest current biomarker) conferred worse survival (HR 2.13, p = 0.002). 66/67 (98.5%) patients analysed revealed telomeric fusions. Nanopore sequencing validated these events, while confirming the presence of microhomology and telomeric deletion. This mutational spectrum is consistent with utilisation of specific DNA repair pathways in mediating telomere fusion. Conclusion This study provides the first mechanistic evidence of dysfunctional telomeres in adult GBMs and suggests TL analysis may provide independent prognostic information. The presence of telomere fusions indicates significant genomic instability and may favour targeted therapies for patients exhibiting this signature.

Background Genomic testing of somatic and germline DNA has transformed cancer care. However, low genetic knowledge among patients may compromise care and health outcomes. Given the rise in genomic testing, we sought to understand patients’ knowledge of their genetic test results. Materials and Methods We conducted a survey‐based study with 85 patients at a comprehensive cancer center. We compared self‐reported recall of (a) having had somatic/germline testing and (b) their specific somatic/germline results to the genomic test results documented in the medical record. Results Approximately 30% of patients did not recall having had testing. Of those who recalled having testing, 44% of patients with pathogenic/likely pathogenic germline mutations and 57% of patients with reported somatic alterations did not accurately recall their specific gene or variant‐level results. Conclusion Given significant knowledge gaps in patients’ recall of genomic testing, there is a critical need to improve patient‐directed education and return‐of‐results strategies., Considering the increase in genomic testing for cancer care, this study aimed to better understand patients’ knowledge of their genetic test results.

Introduction Accurate manoeuvring of the Sylvian fissure borders to provide an optimal operating corridor is vital for minimising retraction. The anatomy of the fissure can pose technical challenges impacting the overall success of the procedure. Yaşargil previously proposed a classification distinguishing four anatomical variants correlating with surgical difficulty. We aim to utilise the open-source Human Connectome Project MRI data set to provide a computational classification of the fissure based on morphology. Method MR images of 2226 hemispheres were included in this study. Morphometrical parameters were designed and refined in MATLAB simulations. Combinations of parameters were analysed using K-Means Clustering. Results Following segmentation and removal of outliers, 1693 fissures remain for analysis. K-Means Clustering revealed three clusters with sulcal bulk displacement measure (deviation of the fissure towards the frontal or temporal lobe) and sulcal distance (width of the fissure) (silhouette score = 0.383). Conclusions Analysis of a large neuroimaging cohort suggested the existence of three weakly separated anatomical variants. We propose that looking at the quantitative scores rather than class allocation would be more informative for preoperative planning.

Understanding multiple challenges that restrict conservation success is a central task of applied ecology, especially when resources are limited and actions are expensive, such as with reintroduction programs. Simultaneous consideration of multiple hypotheses can expedite identification of factors that most limit conservation success. Since 2001, reintroduction of a migratory population of Whooping Cranes (Grus americana) has been under way in eastern North America. Hatching success, however, has been extremely low. In our study area, in and near Necedah National Wildlife Refuge in central Wisconsin, USA, we simultaneously tested 3 hypotheses explaining poor hatching success: harassment of incubating birds by black flies (Simuliidae), effects of captivity, and inexperience of breeders. When black flies were experimentally suppressed, hatching probability doubled. Daily nest survival for Whooping Cranes was strongly and negatively related to an index of black fly abundance, particularly of Simuliu...

Background In germline genetic testing, variants from understudied ancestries have been disproportionately classified as being of uncertain significance. We hypothesized that the rate of variant reclassification likewise differs by ancestry. Methods Nonbenign variants in actionable genes were collected from consenting subjects undergoing genetic testing at two Southern California sites from September 1996 through December 2016. Variant reclassifications were recorded as they were received, until February 2017 or reclassification to benign. Excluding duplicate variants (same ancestry, laboratory, classification), generalized linear models for the hereditary breast cancer genes (BRCA1/2) and other variants investigated whether rate of reclassification differed for seven categories of ancestry compared with non-Hispanic European. Models took into account laboratory, year, gene, sex, and current classification (handled as a time-dependent covariate) and were adjusted for multiple hypothesis testing. Results Among 1483 nonbenign variants, 693 (46.7%) involved BRCA1/2. Overall, 268 (18.1%) variants were reclassified at least once. Few (9.7%) reclassified variants underwent a net upgrade in pathogenicity. For BRCA1/2 variants, reclassification rates varied by ancestry and increased over time, more steeply for ancestries with lower initial rates (African, Ashkenazi, Chinese) than for ancestries whose initial rates were high (Middle Eastern) or similar to non-Hispanic European (non-Chinese Asian, Native American, Hispanic). In contrast, reclassification rates of non-BRCA1/2 variants did not vary over time but were elevated for most minority ancestries except non-Chinese Asian and Native American. Conclusions For nonbenign variants in cancer-related genes, the rates at which reclassifications are issued vary by ancestry in ways that differ between BRCA1/2 and other genes.

Background Glioblastoma is a rapidly fatal brain cancer that exhibits extensive intra- and intertumoral heterogeneity. Improving survival will require the development of personalized treatment strategies that can stratify tumors into subtypes that differ in therapeutic vulnerability and outcomes. Glioblastoma stratification has been hampered by intratumoral heterogeneity, limiting our ability to compare tumors in a consistent manner. Here, we develop methods that mitigate the impact of intratumoral heterogeneity on transcriptomic-based patient stratification. Methods We accessed open-source transcriptional profiles of histological structures from 34 human glioblastomas from the Ivy Glioblastoma Atlas Project. Principal component and correlation network analyses were performed to assess sample inter-relationships. Gene set enrichment analysis was used to identify enriched biological processes and classify glioblastoma subtype. For survival models, Cox proportional hazards regression was utilized. Transcriptional profiles from 156 human glioblastomas were accessed from The Cancer Genome Atlas to externally validate the survival model. Results We showed that intratumoral histologic architecture influences tumor classification when assessing established subtyping and prognostic gene signatures, and that indiscriminate sampling can produce misleading results. We identified the cellular tumor as a glioblastoma structure that can be targeted for transcriptional analysis to more accurately stratify patients by subtype and prognosis. Based on expression from cellular tumor, we created an improved risk stratification gene signature. Conclusions Our results highlight that biomarker performance for diagnostics, prognostics, and prediction of therapeutic response can be improved by analyzing transcriptional profiles in pure cellular tumor, which is a critical step toward developing personalized treatment for glioblastoma.

Objective Clinicians’ ability to use and interpret genetic information depends upon how those data are displayed in electronic health records (EHRs). There is a critical need to develop systems to effectively display genetic information in EHRs and augment clinical decision support (CDS). Materials and Methods The National Institutes of Health (NIH)-sponsored Clinical Sequencing Exploratory Research and Electronic Medical Records & Genomics EHR Working Groups conducted a multiphase, iterative process involving working group discussions and 2 surveys in order to determine how genetic and genomic information are currently displayed in EHRs, envision optimal uses for different types of genetic or genomic information, and prioritize areas for EHR improvement. Results There is substantial heterogeneity in how genetic information enters and is documented in EHR systems. Most institutions indicated that genetic information was displayed in multiple locations in their EHRs. Among surveyed institutions, genetic information enters the EHR through multiple laboratory sources and through clinician notes. For laboratory-based data, the source laboratory was the main determinant of the location of genetic information in the EHR. The highest priority recommendation was to address the need to implement CDS mechanisms and content for decision support for medically actionable genetic information. Conclusion Heterogeneity of genetic information flow and importance of source laboratory, rather than clinical content, as a determinant of information representation are major barriers to using genetic information optimally in patient care. Greater effort to develop interoperable systems to receive and consistently display genetic and/or genomic information and alert clinicians to genomic-dependent improvements to clinical care is recommended.

Motivation: Networks are widely used as structural summaries of biochemical systems. Statistical estimation of networks is usually based on linear or discrete models. However, the dynamics of biochemical systems are generally non-linear, suggesting that suitable non-linear formulations may offer gains with respect to causal network inference and aid in associated prediction problems. Results: We present a general framework for network inference and dynamical prediction using time course data that is rooted in non-linear biochemical kinetics. This is achieved by considering a dynamical system based on a chemical reaction graph with associated kinetic parameters. Both the graph and kinetic parameters are treated as unknown; inference is carried out within a Bayesian framework. This allows prediction of dynamical behavior even when the underlying reaction graph itself is unknown or uncertain. Results, based on (i) data simulated from a mechanistic model of mitogen-activated protein kinase signaling and (ii) phosphoproteomic data from cancer cell lines, demonstrate that non-linear formulations can yield gains in causal network inference and permit dynamical prediction and uncertainty quantification in the challenging setting where the reaction graph is unknown. Availability and implementation: MATLAB R2014a software is available to download from warwick.ac.uk/chrisoates. Contact: c.oates@warwick.ac.uk or sach@mrc-bsu.cam.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online.

Aberrant DNA methylation of CpG islands, CpG island shores and first exons is known to play a key role in the altered gene expression patterns in all human cancers. To date, a systematic study on the effect of DNA methylation on gene expression using high resolution data has not been reported. In this study, we conducted an integrated analysis of MethylCap-sequencing data and Affymetrix gene expression microarray data for 30 breast cancer cell lines representing different breast tumor phenotypes. As well-developed methods for the integrated analysis do not currently exist, we created a series of four different analysis methods. On the computational side, our goal is to develop methylome data analysis protocols for the integrated analysis of DNA methylation and gene expression data on the genome scale. On the cancer biology side, we present comprehensive genome-wide methylome analysis results for differentially methylated regions and their potential effect on gene expression in 30 breast cancer cell lines representing three molecular phenotypes, luminal, basal A and basal B. Our integrated analysis demonstrates that methylation status of different genomic regions may play a key role in establishing transcriptional patterns in molecular subtypes of human breast cancer.

The most bacteria-like mitochondrial genome known is that of the jakobid flagellate Reclinomonas americana NZ. This genome also encodes the largest known gene set among mitochondrial DNAs (mtDNAs), including the RNA subunit of RNase P (transfer RNA processing), a reduced form of transfer–messenger RNA (translational control), and a four-subunit bacteria-like RNA polymerase, which in other eukaryotes is substituted by a nucleus-encoded, single-subunit, phage-like enzyme. Further, protein-coding genes are preceded by potential Shine–Dalgarno translation initiation motifs. Whether similarly ancestral mitochondrial characters also exist in relatives of R. americana NZ is unknown. Here, we report a comparative analysis of nine mtDNAs from five distant jakobid genera: Andalucia, Histiona, Jakoba, Reclinomonas, and Seculamonas. We find that Andalucia godoyi has an even larger mtDNA gene complement than R. americana NZ. The extra genes are rpl35 (a large subunit mitoribosomal protein) and cox15 (involved in cytochrome oxidase assembly), which are nucleus encoded throughout other eukaryotes. Andalucia cox15 is strikingly similar to its homolog in the free-living α-proteobacterium Tistrella mobilis. Similarly, a long, highly conserved gene cluster in jakobid mtDNAs, which is a clear vestige of prokaryotic operons, displays a gene order more closely resembling that in free-living α-proteobacteria than in Rickettsiales species. Although jakobid mtDNAs, overall, are characterized by bacteria-like features, they also display a few remarkably divergent characters, such as 3′-tRNA editing in Seculamonas ecuadoriensis and genome linearization in Jakoba libera. Phylogenetic analysis with mtDNA-encoded proteins strongly supports monophyly of jakobids with Andalucia as the deepest divergence. However, it remains unclear which α-proteobacterial group is the closest mitochondrial relative.

Chlorarachniophytes are unicellular marine algae with plastids (chloroplasts) of secondary endosymbiotic origin. Chlorarachniophyte cells retain the remnant nucleus (nucleomorph) and cytoplasm (periplastidial compartment, PPC) of the green algal endosymbiont from which their plastid was derived. To characterize the diversity of nucleus-encoded proteins targeted to the chlorarachniophyte plastid, nucleomorph, and PPC, we isolated plastid–nucleomorph complexes from the model chlorarachniophyte Bigelowiella natans and subjected them to high-pressure liquid chromatography-tandem mass spectrometry. Our proteomic analysis, the first of its kind for a nucleomorph-bearing alga, resulted in the identification of 324 proteins with 95% confidence. Approximately 50% of these proteins have predicted bipartite leader sequences at their amino termini. Nucleus-encoded proteins make up >90% of the proteins identified. With respect to biological function, plastid-localized light-harvesting proteins were well represented, as were proteins involved in chlorophyll biosynthesis. Phylogenetic analyses revealed that many, but by no means all, of the proteins identified in our proteomic screen are of apparent green algal ancestry, consistent with the inferred evolutionary origin of the plastid and nucleomorph in chlorarachniophytes.

Water was collected from a site on the Susquehanna River in eastern Pennsylvania, where less-than-optimal black fly larval mortality had been occasionally observed after treatment with Bacillus thuringiensis subsp. israelensis de Barjac insecticidal crystalline proteins (Bti ICPs). A series of experiments was conducted with Simulium vittatum Zetterstedt larvae to determine the water related factors responsible for the impaired response to Bti ICPs (Vectobac 12S, strain AM 65-52). Seston in the water impaired the effectiveness of the ICPs, whereas the dissolved substances had no impact on larval mortality. Individual components of the seston then were exposed to the larvae followed by exposure to Bti ICPs. Exposure of larvae to selected minerals and nutritive organic material before ICP exposure resulted in no significant decrease in mortality. Exposure of larvae to silicon dioxide, cellulose, viable diatoms, and purified diatom frustules before Bti ICP exposure resulted in significant reductions in mortality. Exposure of larvae to purified diatom frustules from Cyclotella meneghiniana Kützing resulted in the most severe impairment of mortality after Bti ICP exposure. It is postulated that frustule-induced impairment of feeding behavior is responsible for the impairment of larval mortality.

Relative effects of Beef Quality Assurance (BQA)-related defects in market beef and dairy cows and bulls on selling price at auction was evaluated during 2008. The presence and severity of 23 BQA-related traits were determined during sales in Idaho, California, and Utah. Overall, 18,949 unique lots consisting of 23,479 animals were assessed during 125 dairy sales and 79 beef sales. Mean sale price ± SD (per 45.5 kg) for market beef cows, beef bulls, dairy cows, and dairy bulls was $45.15 ± 9.42, $56.30 ± 9.21, $42.23 ± 12.26, and $55.10 ± 9.07, respectively. When combined, all recorded traits explained 36% of the variation in selling price in beef cows, 35% in beef bulls, 61% in dairy cows, and 56% in dairy bulls. Premiums and discounts were determined in comparison with a "par" or "base" animal. Compared with a base BCS 5 beef cow (on a 9-point beef scale), BCS 1 to 4 cows were discounted (P < 0.0001), whereas premiums (P < 0.05) were estimated for BCS 6 to 8. Compared with a base BCS 3.0 dairy cow (on a 5-point dairy scale), more body condition resulted in a premium (P ≤ 0.001), whereas a less-than-desirable BCS of 2.0 or 2.5 was discounted (P < 0.0001). Emaciated or near-emaciated cows (beef BCS 1 or 2; dairy BCS 1.0 or 1.5) were discounted (P < 0.0001). Compared with base cows weighing 545 to 635 kg, lighter BW beef cows were discounted (P < 0.0001), whereas heavier beef cows received (P < 0.05) a premium. Compared with a base dairy cow weighing 636 to 727 kg, lighter BW cows were discounted (P < 0.0001), whereas heavier cows (727 to 909 kg) received a premium (P < 0.01). Beef and dairy cows with any evidence of lameness were discounted (P < 0.0001). Presence of ocular neoplasia in the precancerous stage discounted (P = 0.05) beef cows and discounted (P < 0.01) dairy cows, whereas at the cancerous stage, it discounted (P < 0.0001) all cows. Hide color influenced (P < 0.0001) selling price in beef cattle but had no effect (P = 0.17) in dairy cows. Animals that were visibly sick were discounted (P < 0.0001). Results suggest that improving BCS and BW, which producers can do at the farm or ranch level, positively affects sale price. Furthermore, animals that are visibly sick or have a defect associated with a possible antibiotic risk will be discounted. Ultimately, animals with minor quality defects should be sold in a timely manner before the defect advances and the discount increases.

A survey was conducted to quantify incidence of Beef Quality Assurance (BQA)-related defects in market beef and dairy cows and bulls selling at auction during 2 seasons in 2008. Twenty-three BQA-related traits were evaluated by 9 trained personnel during sales at 10 livestock auction markets in Idaho (n = 5; beef and dairy), California, (n = 4; dairy only), and Utah (n = 1; beef and dairy). Overall, 18,949 unique lots (8,213 beef cows, 1,036 beef bulls, 9,177 dairy cows, and 523 dairy bulls,) consisting of 23,479 animals (9,299 beef cows, 1,091 beef bulls, 12,429 dairy cows, and 660 dairy bulls) were evaluated during 125 sales (64 spring, 61 fall) for dairy and 79 sales (40 spring, 39 fall) for beef. The majority of market beef cows and bulls (60.9 and 71.3%, respectively) were predominantly black-hided, and the Holstein hide pattern was observed in 95.4 and 93.6% of market dairy cows and bulls, respectively. Market cattle weighed 548 ± 103.6 kg (beef cows), 751 ± 176.1 kg (beef bulls), 658 ± 129.7 kg (dairy cows), and 731 ± 150.8 kg (dairy bulls). Most beef cows (79.6%) weighed 455 to 726 kg, and most beef bulls (73.8%) weighed 545 to 954 kg, respectively. Among market beef cattle, 16.0% of cows and 14.5% of bulls weighed less than 455 and 545 kg, respectively, and 63.7% of dairy cows and 81.5% of dairy bulls weighed 545 to 817 kg or 545 to 954 kg, respectively. However, 19.5% of dairy cows and 13.1% of dairy bulls weighed less than 545 kg. Mean BCS for beef cattle (9-point scale) was 4.7 ± 1.2 (cows) and 5.3 ± 0.9 (bulls), and for dairy cattle (5-point scale) was 2.6 ± 0.8 (cows) and 2.9 ± 0.6 (bulls). Some 16.5% of beef cows and 4.1% of beef bulls had a BCS of 1 to 3, whereas 34.8% of dairy cows and 10.4% of dairy bulls had a BCS of 2 or less. Emaciation (beef BCS = 1, dairy BCS = 1.0) or near-emaciation (beef BCS = 2, dairy BCS = 1.5) was observed in 13.3% of dairy cows and 3.9% of beef cows. Among beef cattle, 15.1% of cows and 15.4% of bulls were considered lame. In contrast, 44.7% of dairy cows and 26.1% of dairy bulls were lame. Ocular neoplasia (cancer eye) was observed in only 0.6% of beef cows, 0.3% of beef bulls, 0.3% of dairy cows, and 0.0% of dairy bulls. However, among animals with ocular neoplasia, it was cancerous in 34.4% of beef bulls, 48.0% of dairy cows, and 73.3% of beef cows. In conclusion, numerous quality defects are present in market beef and dairy cattle selling at auction in the Western United States, which could influence their value at auction.

Motivation: Molecular association of phenotypic responses is an important step in hypothesis generation and for initiating design of new experiments. Current practices for associating gene expression data with multidimensional phenotypic data are typically (i) performed one-to-one, i.e. each gene is examined independently with a phenotypic index and (ii) tested with one stress condition at a time, i.e. different perturbations are analyzed separately. As a result, the complex coordination among the genes responsible for a phenotypic profile is potentially lost. More importantly, univariate analysis can potentially hide new insights into common mechanism of response. Results: In this article, we propose a sparse, multitask regression model together with co-clustering analysis to explore the intrinsic grouping in associating the gene expression with phenotypic signatures. The global structure of association is captured by learning an intrinsic template that is shared among experimental conditions, with local perturbations introduced to integrate effects of therapeutic agents. We demonstrate the performance of our approach on both synthetic and experimental data. Synthetic data reveal that the multi-task regression has a superior reduction in the regression error when compared with traditional L1-and L2-regularized regression. On the other hand, experiments with cell cycle inhibitors over a panel of 14 breast cancer cell lines demonstrate the relevance of the computed molecular predictors with the cell cycle machinery, as well as the identification of hidden variables that are not captured by the baseline regression analysis. Accordingly, the system has identified CLCA2 as a hidden transcript and as a common mechanism of response for two therapeutic agents of CI-1040 and Iressa, which are currently in clinical use. Contact: b_parvin@lbl.gov

Biting flies have been suggested as mechanical vectors of vesicular stomatitis New Jersey Virus (family Rhabdoviridae, genus Vesiculovirus, VSNJV) in livestock populations during epidemic outbreaks in the western United States. We conducted a proof-of-concept study to determine whether biting flies could mechanically transmit VSNJV to livestock by using a black fly, Simulium vittatum Zetterstedt (Diptera: Simuliidae), domestic swine, Sus scrofa L., model. Black flies mechanically transmitted VSNJV to a naive host after interrupted feeding on a vesicular lesion on a previously infected host. Transmission resulted in clinical disease in the naive host. This is the first demonstration of mechanical transmission of VSNJV to livestock by insects.

Vesicular stomatitis New Jersey virus (VSNJV) is an insect-transmitted Rhabdovirus causing vesicular disease in domestic livestock including cattle, horses, and pigs. Natural transmission during epidemics remains poorly understood, particularly in cattle, one of the most affected species during outbreaks. This study reports the first successful transmission of VSNJV to cattle by insect bite resulting in clinical disease. When infected black flies (Simulium vittatum Zetterstedt) fed at sites where VS lesions are usually observed (mouth, nostrils, and foot coronary band), infection occurred, characterized by local viral replication, vesicular lesions, and high neutralizing antibody titers (1: 256). Viral RNA was detected up to 9 d postinfection in tissues collected during necropsy from lesion sites and lymph nodes draining those sites. Interestingly, when flies were allowed to feed on flank or neck skin, viral replication was poor, lesions were not observed, and low levels of neutralizing antibodies (range, 1:8-1:32) developed. Viremia was never observed in any of the animals and infectious virus was not recovered from tissues on necropsies performed between 8 and 27 d postinfection. Demonstration that VSNJV transmission to cattle by infected black flies can result in clinical disease contributes to a better understanding of the epidemiology and potential prevention and control methods for this important disease.

Fresh Juice Incorporated (FJI) is in the process of determining whether it should launch a new fruit juice, Genetically Enhanced (GE) Juice. The GE Juice meets consumers' demands for a tasty, nutritious product and it would be the first new juice product in the last fifteen years. Before FJI decides to launch GE Juice, it must analyze the uncertainty surrounding market size, market share, and price of GE Juice. Finally, if FJI decides to launch GE Juice, then they must decide if they will bottle the juice themselves or outsource this process. This case teaches students how to discuss the strategic implications of launching a new product and develop a net present value and financial feasibility simulation model given limited information. Copyright 2008, Oxford University Press.

Microscopic, unicellular, free-living green algae are found in desert microbiotic crusts worldwide. Although morphologically simple, green algae in desert crusts have recently been found to be extraordinarily diverse, with membership spanning five green algal classes and encompassing many taxa new to science. This overview explores this remarkable diversity and its potential to lead to new perspectives on the diversity and evolution of green plants. Molecular systematic and physiological data gathered from desert taxa demonstrate that these algae are long-term members of desert communities, not transient visitors from aquatic habitats. Variations in desiccation tolerance and photophysiology among these algae include diverse evolutionary innovations that developed under selective pressures in the desert. Combined with the single embryophyte lineage to which more familiar terrestrial green plants belong, multiple desert green algal lineages provide independent evolutionary units that may enhance understanding of the evolution and ecology of eukaryotic photosynthetic life on land.

Box C/D ribonucleoprotein (RNP) particles mediate O2′-methylation of rRNA and other cellular RNA species. In higher eukaryotic taxa, these RNPs are more complex than their archaeal counterparts, containing four core protein components (Snu13p, Nop56p, Nop58p and fibrillarin) compared with three in Archaea. This increase in complexity raises questions about the evolutionary emergence of the eukaryote-specific proteins and structural conservation in these RNPs throughout the eukaryotic domain. In protists, the primarily unicellular organisms comprising the bulk of eukaryotic diversity, the protein composition of box C/D RNPs has not yet been extensively explored. This study describes the complete gene, cDNA and protein sequences of the fibrillarin homolog from the protozoon Euglena gracilis, the first such information to be obtained for a nucleolus-localized protein in this organism. The E.gracilis fibrillarin gene contains a mixture of intron types exhibiting markedly different sizes. In contrast to most other E.gracilis mRNAs characterized to date, the fibrillarin mRNA lacks a spliced leader (SL) sequence. The predicted fibrillarin protein sequence itself is unusual in that it contains a glycine-lysine (GK)-rich domain at its N-terminus rather than the glycine-arginine-rich (GAR) domain found in most other eukaryotic fibrillarins. In an evolutionarily diverse collection of protists that includes E.gracilis, we have also identified putative homologs of the other core protein components of box C/D RNPs, thereby providing evidence that the protein composition seen in the higher eukaryotic complexes was established very early in eukaryotic cell evolution.

The role of hematophagous arthropods in vesicular stomatitis virus (New Jersey serotype; VSV-NJ) transmission during epizootics has remained unclear for decades in part because it has never been shown that clinical or subclinical disease in a livestock host results from the bite of an infected insect. In this study, we investigated the ability of VSV-NJ-infected black flies (Simulium vittatum Zetterstedt) to transmit the virus to domestic swine, Sus scrofa L. Experimental evidence presented here clearly demonstrates that VSV-NJ was transmitted from black flies to the swine. Transmission was confirmed by seroconversion or by the presence of clinical vesicular stomatitis followed by seroconversion. Our results represent the first report of clinical vesicular stomatitis in a livestock host after virus transmission by an insect.

The ability to immunologically link microscopic tags to specific proteins has produced major advances in all forms of microscopy. Initially researchers needed to make and label their own antibodies. This required a good working knowledge of immunology. Today's labeled antibodies are readily available from a variety of vendors. Although this has greatly facilitated the expansion of immunolabeling techniques, it has also led to their use by researchers with little or no background in immunology.

Microscopy has played a critical role in first identifying and then defining the role of lysosomes in formation of atherosclerotic foam cells. We review the evidence implicating lysosomal lipid accumulation as a factor in the pathogenesis of atherosclerosis with reference to the role of microscopy. In addition, we explore mechanisms by which lysosomal lipid engorgement occurs. Low density lipoproteins which have become modified are the major source of lipid for foam cell formation. These altered lipoproteins are taken into the cell via receptor-mediated endocytosis and delivered to lysosomes. Under normal conditions, lipids from these lipoproteins are metabolized and do not accumulate in lysosomes. In the atherosclerotic foam cell, this normal metabolism is inhibited so that cholesterol and cholesteryl esters accumulate in lysosomes. Studies of cultured cells incubated with modified lipoproteins suggests this abnormal metabolism occurs in two steps. Initially, hydrolysis of lipoprotein cholesteryl esters occurs normally, but the resultant free cholesterol cannot exit the lysosome. Further lysosomal cholesterol accumulation inhibits hydrolysis, producing a mixture of cholesterol and cholesteryl esters within swollen lysosomes. Various lipoprotein modifications can produce this lysosomal engorgement in vitro and it remains to be seen which modifications are most important in vivo.

Laser capture microdissection uses a microscope to identify specific cells for microdissection and then a laser-sensitive plastic to capture and remove the cells from their substrate. This efficient capture method was originally developed to capture cells for genetic analysis. However, it has also been used to capture cells for proteonomic analysis. In this article, we extend the uses of laser-capture microdissection by reporting a method for preparing captured cells for ultrastructural analysis by transmission electron microscopy. Cells prepared by our methodology show good fine structure preservation and are easily sectioned by standard ultramicrotomy.

The aim of this study was to determine whether or not the use of medical pre-treatment of the endometrium improves the outcome of transcervical resection of the endometrium with regards to long-term operative outcome, histological findings and patient satisfaction. A prospective randomized trial comparing three endometrial pre-treatment agents (danazol, medroxyprogesterone acetate or nafarelin) with no pre-treatment was conducted. The main outcome measures were: (i) thickness of the endometrium and myometrium resected; (ii) histological stage of the endometrium at the time of operation; (iii) the presence or absence of menses and (iv) patient satisfaction 1 year post-operatively. Of the three pre-treatments studied, danazol produced a lower median endometrial thickness than the control, showed the greatest ability to induce atrophy of the endometrial glands and stroma (not statistically significant) and produced the highest rate of amenorrhoea (not different to the control). Danazol and nafarelin produced significantly lower median endometrial thickness than no pre-treatment. There were, however, no significant differences in the rates of amenorrhoea in any of the pre-treatment groups compared with that in the control group. No improvement in clinical outcome or patient satisfaction is conferred by the use of medical pre-treatments if transcervical resection of the endometrium is performed in the proliferative phase of the menstrual cycle.

Most objects in our world are 3-dimensionai (3-D), and this is certainly true of cellular ultrastructure. The challenge in microscopy has always been how to analyze this 3-D information. Traditional thin-section microscopy has a minimal ability to view 3-D structure, because it is limited to viewing thin, almost twodimensional (2-D) planes taken from the specimen. The development of high and intermediate voltage electron microscopes (HVEM and IVEM) provides the ability to investigate the ultrastructure of thick biological samples—allowing a unique view of the 3-D interrelationships of cells and organelles.

Genome-wide analysis techniques such as chromosome painting, comparative genomic hybridization, representational difference analysis, restriction landmark genome scanning and high-throughput analysis of LOH are now accelerating high-resolution genome aberration localization in human tumors. These techniques are complemented by procedures for detection of differentially expressed genes such as differential display, nucleic acid subtraction, serial analysis of gene expression and expression microarray analysis. These efforts are enabled by work from the human genome program in physical map development, cDNA library production/sequencing and in genome sequencing. This review covers several commonly used large-scale genome and gene expression analysis techniques, outlines genomic approaches to gene discovery and summarizes information that has come from large-scale analyses of human solid tumors.

The mitochondrial DNA (mtDNA) of Porphyra purpurea , a circular-mapping genome of 36,753 bp, has been completely sequenced. A total of 57 densely packed genes has been identified, including the basic set typically found in animals and fungi, as well as seven genes characteristic of protist and plant mtDNAs and specifying ribosomal proteins and subunits of succinate:ubiquinone oxido-reductase. The mitochondrial large subunit rRNA gene contains two group II introns that are extraordinarily similar to those found in the cyanobacterium Calothrix sp, suggesting a recent lateral intron transfer between a bacterial and a mitochondrial genome. Notable features of P. purpurea mtDNA include the presence of two 291-bp inverted repeats that likely mediate homologous recombination, resulting in genome rearrangement, and of numerous sequence polymorphisms in the coding and intergenic regions. Comparative analysis of red algal mitochondrial genomes from five different, evolutionarily distant orders reveals that rhodophyte mtDNAs are unusually uniform in size and gene order. Finally, phylogenetic analyses provide strong evidence that red algae share a common ancestry with green algae and plants.

In this article we review recent advances in our understanding of the biochemistry and evolution of the mitochondrial transcriptional apparatus, with emphasis on flowering plants (angiosperms). In the majority of eukaryotes studied to date, including plants, the mitochondrial RNA (mtRNA) polymerase is a single-polypeptide, bacteriophage T3/T7-like enzyme, encoded in the nucleus. While the evolutionary origin of the T3/T7 RNA polymerase itself remains an enigma, the emergence of a phagelike mtRNA polymerase seems to have closely paralleled the appearance of mitochondria within the eukaryotic cell. In angiosperms, several groups have discovered a second phagelike RNA polymerase gene, evidently arising via duplication of a preexisting gene encoding the mtRNA polymerase. This second gene specifies a phagelike activity that functions in the chloroplast, in addition to the eubacteria-like RNA polymerase encoded by the chloroplast genome. The chloroplast situation appears to recapitulate an early stage in the evolution of the mitochondrial transcriptional machinery: that is, appearance of a phagelike enzyme that eventually displaced a multisubunit, eubacteria-like RNA polymerase originally encoded in the protomitochondrial genome. In angiosperms, candidate transcription factors (promoter-specific DNA-binding proteins) have been characterized in wheat (63 kDa) and pea (34 and 44 kDa). This difference between monocotyledonous and dicotyledonous plants may reflect known differences in the promoter sequences recognized by the mtRNA polymerase in the two angiosperm groups.

Helper T cells are classified into Th1 and Th2 subsets based on their profiles of cytokine production. Th1 cells are involved in cell-mediated immunity, whereas Th2 cells induce humoral responses. Selective recruitment of these two subsets depends on specific adhesion molecules and specific chemoattractants. Here, we demonstrate that the T cell-directed CC chemokine thymus and activation-regulated chemokine (TARC) was abundantly produced by monocytes treated with granulocyte macrophage colony stimulating factor (GM-CSF) or IL-3, especially in the presence of IL-4 and by dendritic cells derived from monocytes cultured with GM-CSF + IL-4. The receptor for TARC and another macrophage/dendritic cell-derived CC chemokine macrophage-derived chemokine (MDC) is CCR4, a G protein-coupled receptor. CCR4 was found to be expressed on approximately 20% of adult peripheral blood effector/memory CD4+ T cells. T cells attracted by TARC and MDC generated cell lines predominantly producing Th2-type cytokines, IL-4 and IL-5. Fractionated CCR4+ cells but not CCR4- cells also selectively gave rise to Th2-type cell lines. When naive CD4+ T cells from adult peripheral blood were polarized in vitro, Th2-type cells selectively expressed CCR4 and vigorously migrated toward TARC and MDC. Taken together, CCR4 is selectively expressed on Th2-type T cells and antigen-presenting cells may recruit Th2 cells expressing CCR4 by producing TARC and MDC in Th2-dominant conditions.

High throughput partial sequencing of randomly selected cDNA clones has proven to be a powerful tool for examining the relative abundance of mRNAs and for the identification of novel gene products. Because of the important role played by macrophages in immune and inflammatory responses, we sequenced over 3000 randomly selected cDNA clones from a human macrophage library. These sequences represent a molecular inventory of mRNAs from macrophages and provide a catalog of highly expressed transcripts. Two of the most abundant clones encode recently identified CC chemokines. Macrophage-derived chemokine (MDC) plays a complex role in immunoregulation and is a potent chemoattractant for dendritic cells, T cells, and natural killer cells. The chemokine receptor CCR4 binds MDC with high affinity and also responds by calcium flux and chemotaxis. CCR4 has been shown to be expressed by Th2 type T cells. Recent studies also implicate MDC as a major component of the host defense against human immunodeficiency virus. J. Leukoc. Biol. 64: 49–54; 1998.

Although the collection of completely sequenced mitochondrial genomes is expanding rapidly, only recently has a phylogenetically broad representation of mtDNA sequences from protists (mostly unicellular eukaryotes) become available. This review surveys the 23 complete protist mtDNA sequences that have been determined to date, commenting on such aspects as mitochondrial genome structure, gene content, ribosomal RNA, introns, transfer RNAs and the genetic code and phylogenetic implications. We also illustrate the utility of a comparative genomics approach to gene identification by providing evidence that orfB in plant and protist mtDNAs is the homolog of atp8 , the gene in animal and fungal mtDNA that encodes subunit 8 of the F0portion of mitochondrial ATP synthase. Although several protist mtDNAs, like those of animals and most fungi, are seen to be highly derived, others appear to be have retained a number of features of the ancestral, proto-mitochondrial genome. Some of these ancestral features are also shared with plant mtDNA, although the latter have evidently expanded considerably in size, if not in gene content, in the course of evolution. Comparative analysis of protist mtDNAs is providing a new perspective on mtDNA evolution: how the original mitochondrial genome was organized, what genes it contained, and in what ways it must have changed in different eukaryotic phyla.