Social Workers' Perspectives on Socially Isolated Older Adults Living with a Robot Companion.

Addressing depression and social isolation among solo-living older adults in South Korea requires a multi-faceted approach. This study aims to explore the advantages and obstacles of a robotic elder care program by analyzing social workers' viewpoints regarding interactions between older adults and a companion robot named Hyodol. Through the purposive sampling method, we conducted comprehensive interviews with 10 social workers who are pioneering the integration of robots in elder care. The study participants observed and compared the demographic characteristics of users and non-users, elucidated usage patterns, described the roles of robot companions, and shared remarkable instances. Overall, the experiences of these social workers underscored the positive influence of Hyodol in offering companionship, care, and emotional support, especially in circumstances where clients experienced isolation or lacked regular family interactions. In addition, social workers' perspectives hold significant value in comprehending the genuine effects of anthropomorphism - the inclination to attribute human-like qualities and emotions to robots. Our investigation revealed the existence of two distinct modes of personification among older adults, which we have labeled as "imagination" and "projection."

Who is behind the robot? The role of public social workers in implementing robotic eldercare program in South Korea.

A companion robot named Hyodol is a digital technology implemented for eldercare in South Korea. Drawing insights from semi-structured interviews with public social workers actively involved in the Hyodol care program, this study explores how social workers contribute to the success of the robotic care program. Throughout the phases of selecting potential users, introducing older adults to the robot, and maintaining the robotic program, the practical wisdom of social workers plays an important role. Despite the increased workload in case management and the emotional labor associated with navigating the care system, these pioneering social workers maintained high morale to adopt the robotic care system. By shedding light on the specific roles of social workers, this study contributes to a deeper understanding of the intricate dynamics that underlie successful robotic eldercare.

[Self-Management Experiences of the Adolescents with Chronic Kidney Disease].

PURPOSE: The aim of this study was to develop a substantive theory on self-management conducted by the adolescents with chronic kidney disease from their lived experience. METHODS: Data was collected through in-depth interviews from May to December in 2015 with thirteen adolescents with chronic kidney disease. The data collected were analyzed on the basis of Strauss and Corbin's grounded theory. RESULTS: The core of the category found in this study was "overcoming the unstable sense of self-control and integrating disease experience into their life". The causal conditions triggering the central phenomenon were "restriction in daily life" and "manifestation and aggravation of symptom". The central phenomenon in the experience of self-management within the adolescents with chronic kidney disease was "unstable sense of self control". The intervening condition for unstable self control were "micro system support" and "motivational resources". This study found that the adolescents with chronic kidney disease followed a series of strategies when they faced the central phenomenon, including; passive coping, reappraisal of illness, active coping, compliance with treatment, controlling physical activity, and adjusting school life. With these strategic approaches, the adolescents with chronic kidney disease could maintain their active lifestyles and achieve their health behaviors. The process of self-management by these adolescents passed through four phases; limited experience caused by diseases, effort for normalization, reorganizing their daily lives, and integration with daily lives and self-management. CONCLUSION: This Study explored the process and experience of self-management of adolescents with chronic kidney disease. These findings can be used for basis for developing substantive theory and nursing intervention strategy for adolescents with chronic kidney diseases.

Evaluation of a genomic classifier in radical prostatectomy patients with lymph node metastasis.

OBJECTIVE: To evaluate the performance of the Decipher test in predicting lymph node invasion (LNI) on radical prostatectomy (RP) specimens. METHODS: We identified 1,987 consecutive patients with RP who received the Decipher test between February and August 2015 (contemporary cohort). In the contemporary cohort, only the Decipher score from RP specimens was available for analysis. In addition, we identified a consecutive cohort of patients treated with RP between 2006 and 2012 at the University of California, San Diego, with LNI upon pathologic examination (retrospective cohort). The retrospective cohort yielded seven, 22, and 18 tissue specimens from prostate biopsy, RP, and lymph nodes (LNs) for individual patients, respectively. Univariable and multivariable logistic regression analyses were used to evaluate the performance of Decipher in the contemporary cohort with LNI as the endpoint. In the retrospective cohort, concordance of risk groups was assessed using validated cut-points for low (<0.45), intermediate (0.45-0.60), and high (>0.60) Decipher scores. RESULTS: In the contemporary cohort, 51 (2.6%) patients had LNI. Decipher had an odds ratio of 1.73 (95% confidence interval, 1.46-2.05) and 1.42 (95% confidence interval, 1.19-1.7) per 10% increase in score on univariable and multivariable (adjusting for pathologic Gleason score, extraprostatic extension, and seminal vesicle invasion), respectively. No significant difference in the clinical and pathologic characteristics between the LN positive patients of contemporary and retrospective cohorts was observed (all P>0.05). Accordingly, among LN-positive patients in the contemporary cohort and retrospective cohort, 80% and 77% had Decipher high risk scores (P=1). In the retrospective cohort, prostate biopsy cores with the highest Gleason grade and percentage of tumor involvement had 86% Decipher risk concordance with both RP and LN specimens. CONCLUSION: Decipher scores were highly concordant between pre- and post-surgical specimens. Further, Decipher scores from RP tissue were predictive of LNI at RP. If validated in a larger cohort of prostate biopsy specimens for prediction of adverse pathology at RP, Decipher may be useful for improved pre-operative staging.

Application of a Clinical Whole-Transcriptome Assay for Staging and Prognosis of Prostate Cancer Diagnosed in Needle Core Biopsy Specimens.

Molecular and genomic analysis of microscopic quantities of tumor from formalin-fixed, paraffin-embedded biopsy specimens has many unique challenges. Herein, we evaluated the feasibility of obtaining transcriptome-wide RNA expression to measure prognostic classifiers in diagnostic prostate needle core biopsy specimens. One-hundred fifty-eight samples from diagnostic needle core biopsy specimens (BX) and radical prostatectomies (RPs) were collected from 33 patients at three hospitals; each patient provided up to six tumor and benign samples. Genome-wide transcriptomic profiles were generated using Affymetrix Human Exon arrays for comparison of gene expression alterations and prognostic signatures between the BX and RP samples. A sufficient amount of RNA (>100 ng) was obtained from all RP specimens (n = 77) and from 72 of 81 of BX specimens. Of transcriptomic features detected in RP, 95% were detectable in BX tissues and demonstrated a high correlation (r = 0.96). Likewise, an expression signature pattern validated on RPs (Decipher prognostic test) showed correlation between BX and RP (r = 0.70). Of matched BX and RP pairs, 25% showed discordant molecular subtypes. Genome-wide exon arrays yielded data of comparable quality from biopsy and RP tissues. The high concordance of tumor-associated gene expression changes between BX and RP samples provides evidence for the adequate performance of the assay platform with samples from prostate needle biopsy specimens with limited tumor volume.

Impact of a Genomic Classifier of Metastatic Risk on Postprostatectomy Treatment Recommendations by Radiation Oncologists and Urologists.

OBJECTIVE: To evaluate how a genomic classifier (GC) that predicts the risk of metastasis after prostatectomy would impact adjuvant treatment recommendations made by radiation oncologists and urologists. The 2 specialties often disagree about postprostatectomy adjuvant treatment recommendations. MATERIALS AND METHODS: Twenty-six radiation oncologists and 20 urologists with genitourinary oncology expertise reviewed de-identified clinical results from 11 patients after radical prostatectomy and made adjuvant treatment recommendations. The same cases were later randomized and reassigned, and treatment recommendations were made using the clinical information and GC test results together. RESULTS: Using clinical information alone, observation was recommended in 42% of decisions made by urologists vs 23% by radiation oncologists (P < .0001). The GC test results altered 35% and 45% of treatment recommendations made by radiation oncologists and urologists, respectively. Multivariate analysis showed GC risk was the strongest factor influencing treatment recommendations by both specialties, with an adjusted odds ratio of 4.17 (95% confidence interval [CI], 2.26-7.70) and 6.51 (95% CI, 4.29-9.88) for radiation oncologists and urologists, respectively. GC results indicating high metastatic risk resulted in intensification of treatment, whereas low metastatic risk resulted in less aggressive recommendations. The GC results increased interdisciplinary agreement in treatment recommendations, as the odds of a recommendation for adjuvant treatment by urologists vs radiation oncologists increased from 0.27 (95% CI, 0.17-0.44) to 0.46 (95% CI, 0.29-0.75) after results of the GC test were available. CONCLUSION: The GC test significantly influenced adjuvant postprostatectomy treatment recommendations, reduced disagreement between radiation oncologists and urologists, and has the potential to enhance personalization of postprostatectomy care.

Evolving transcriptomic fingerprint based on genome-wide data as prognostic tools in prostate cancer.

BACKGROUND INFORMATION: Prostate cancer (PCa) is a common disease but only a small subset of patients are at risk of developing metastasis and lethal disease, and identifying which patients will progress is challenging because of the heterogeneity underlying tumour progression. Understanding this heterogeneity at the molecular level and the resulting clinical impact is a critical step necessary for risk stratification. Defining genomic fingerprint elucidates molecular variation and may improve PCa risk stratification, providing more accurate prognostic information of tumour aggressiveness (or lethality) for prognostic biomarker development. Therefore, we explored transcriptomic differences between patients with indolent disease outcome and patients who developed metastasis post-radical prostatectomy using genome-wide expression data in the post radical prostatectomy clinical space before metastatic spread. RESULTS: Based on differential expression analysis, patients with adverse pathological findings who are at higher risk of developing metastasis have a distinct transcriptomic fingerprint that can be detected on surgically removed prostate specimens several years before metastasis detection. Nearly half of the transcriptomic fingerprint features were non-coding RNA highlighting their pivotal role in PCa progression. Protein-coding RNA features in the fingerprint are involved in multiple pathways including cell cycle, chromosome structure maintenance and cytoskeleton organisation. The metastatic transcriptomic fingerprint was determined in independent cohorts verifying the association between the fingerprint and metastatic patients. Further, the fingerprint was confirmed in metastasis lesions demonstrating that the fingerprint represents early metastatic transcriptomic changes, suggesting its utility as a prognostic tool to predict metastasis and provide clinical value in the early radical prostatectomy setting. CONCLUSIONS: Here, we show that transcriptomic patterns of metastatic PCa exist that can be detected early after radical prostatectomy. This metastatic fingerprint has potential prognostic ability that can impact PCa treatment management potentially circumventing the requirements for unnecessary therapies.

Case management for medical aid beneficiaries in Korea: findings from case-control study.

PURPOSE: This study was to evaluate effects of case management provided for 7 months for medical aid in Korea. METHODS: This study was a retrospective comparative study using secondary data analysis. Data from two pre-existing survey were reanalyzed. The data were collected through door to-door interviews using the structured questionnaire. For the medical service use, claims data from the Korea National Health Insurance Corporation was used. Subjects were 73 in the intervention group and 118 in the control group. RESULTS: There was no significant change in the intervention group in self-care ability (p = .296), medication adherence (p = .194) or quality of life (p = .903) compared to those of the control group. For hospital visiting days, it appeared to decrease in the intervention group (p = .038) but with no significant difference from that of the control group (p = .157). Neither were there significant differences in medical expenditures (p = .605). CONCLUSION: Although the effect of case management in this study appeared extremely limited, the short intervention period and characteristics of the medical aid beneficiaries and the limit of controlling only the demand side were discussed as factors to be considered. Nurses have been carrying out professional roles in case management in Korea. However more efforts are needed to develop case management as an area for nursing specialization.

Novel multivariate methods for integration of genomics and proteomics data: applications in a kidney transplant rejection study.

Multi-omics research is a key ingredient of data-intensive life sciences research, permitting measurement of biological molecules at different functional levels in the same individual. For a complete picture at the biological systems level, appropriate statistical techniques must however be developed to integrate different 'omics' data sets (e.g., genomics and proteomics). We report here multivariate projection-based analyses approaches to genomics and proteomics data sets, using the case study of and applications to observations in kidney transplant patients who experienced an acute rejection event (n=20) versus non-rejecting controls (n=20). In this data sets, we show how these novel methodologies might serve as promising tools for dimension reduction and selection of relevant features for different analytical frameworks. Unsupervised analyses highlighted the importance of post transplant time-of-rejection, while supervised analyses identified gene and protein signatures that together predicted rejection status with little time effect. The selected genes are part of biological pathways that are representative of immune responses. Gene enrichment profiles revealed increases in innate immune responses and neutrophil activities and a depletion of T lymphocyte related processes in rejection samples as compared to controls. In all, this article offers candidate biomarkers for future detection and monitoring of acute kidney transplant rejection, as well as ways forward for methodological advances to better harness multi-omics data sets.

Variation in RNA-Seq transcriptome profiles of peripheral whole blood from healthy individuals with and without globin depletion.

BACKGROUND: The molecular profile of circulating blood can reflect physiological and pathological events occurring in other tissues and organs of the body and delivers a comprehensive view of the status of the immune system. Blood has been useful in studying the pathobiology of many diseases. It is accessible and easily collected making it ideally suited to the development of diagnostic biomarker tests. The blood transcriptome has a high complement of globin RNA that could potentially saturate next-generation sequencing platforms, masking lower abundance transcripts. Methods to deplete globin mRNA are available, but their effect has not been comprehensively studied in peripheral whole blood RNA-Seq data. In this study we aimed to assess technical variability associated with globin depletion in addition to assessing general technical variability in RNA-Seq from whole blood derived samples. RESULTS: We compared technical and biological replicates having undergone globin depletion or not and found that the experimental globin depletion protocol employed removed approximately 80% of globin transcripts, improved the correlation of technical replicates, allowed for reliable detection of thousands of additional transcripts and generally increased transcript abundance measures. Differential expression analysis revealed thousands of genes significantly up-regulated as a result of globin depletion. In addition, globin depletion resulted in the down-regulation of genes involved in both iron and zinc metal ion bonding. CONCLUSIONS: Globin depletion appears to meaningfully improve the quality of peripheral whole blood RNA-Seq data, and may improve our ability to detect true biological variation. Some concerns remain, however. Key amongst them the significant reduction in RNA yields following globin depletion. More generally, our investigation of technical and biological variation with and without globin depletion finds that high-throughput sequencing by RNA-Seq is highly reproducible within a large dynamic range of detection and provides an accurate estimation of RNA concentration in peripheral whole blood. High-throughput sequencing is thus a promising technology for whole blood transcriptomics and biomarker discovery.

Longitudinal analysis of whole blood transcriptomes to explore molecular signatures associated with acute renal allograft rejection.

In this study, we explored a time course of peripheral whole blood transcriptomes from kidney transplantation patients who either experienced an acute rejection episode or did not in order to better delineate the immunological and biological processes measureable in blood leukocytes that are associated with acute renal allograft rejection. Using microarrays, we generated gene expression data from 24 acute rejectors and 24 nonrejectors. We filtered the data to obtain the most unambiguous and robustly expressing probe sets and selected a subset of patients with the clearest phenotype. We then performed a data-driven exploratory analysis using data reduction and differential gene expression analysis tools in order to reveal gene expression signatures associated with acute allograft rejection. Using a template-matching algorithm, we then expanded our analysis to include time course data, identifying genes whose expression is modulated leading up to acute rejection. We have identified molecular phenotypes associated with acute renal allograft rejection, including a significantly upregulated signature of neutrophil activation and accumulation following transplant surgery that is common to both acute rejectors and nonrejectors. Our analysis shows that this expression signature appears to stabilize over time in nonrejectors but persists in patients who go on to reject the transplanted organ. In addition, we describe an expression signature characteristic of lymphocyte activity and proliferation. This lymphocyte signature is significantly downregulated in both acute rejectors and nonrejectors following surgery; however, patients who go on to reject the organ show a persistent downregulation of this signature relative to the neutrophil signature.

Gene expression profiling of oxidative stress response of C. elegans aging defective AMPK mutants using massively parallel transcriptome sequencing.

BACKGROUND: A strong association between stress resistance and longevity in multicellular organisms has been established as many mutations that extend lifespan also show increased resistance to stress. AAK-2, the C. elegans homolog of an alpha subunit of AMP-activated protein kinase (AMPK) is an intracellular fuel sensor that regulates cellular energy homeostasis and functions in stress resistance and lifespan extension. FINDINGS: Here, we investigated global transcriptional responses of aak-2 mutants to oxidative stress and in turn identified potential downstream targets of AAK-2 involved in stress resistance in C. elegans. We employed massively parallel Illumina sequencing technology and performed comprehensive comparative transcriptome analysis. Specifically, we compared the transcriptomes of aak-2 and wild type animals under normal conditions and conditions of induced oxidative stress. This research has presented a snapshot of genome-wide transcriptional activities that take place in C. elegans in response to oxidative stress both in the presence and absence of AAK-2. CONCLUSIONS: The analysis presented in this study has enabled us to identify potential genes involved in stress resistance that may be either directly or indirectly under the control of AAK-2. Furthermore, we have extended our current knowledge of general defense responses of C. elegans against oxidative stress supporting the function for AAK-2 in inhibition of biosynthetic processes, especially lipid synthesis, under oxidative stress and transcriptional regulation of genes involved in reproductive processes.

Transcriptome analysis for Caenorhabditis elegans based on novel expressed sequence tags.

BACKGROUND: We have applied a high-throughput pyrosequencing technology for transcriptome profiling of Caenorhabditis elegans in its first larval stage. Using this approach, we have generated a large amount of data for expressed sequence tags, which provides an opportunity for the discovery of putative novel transcripts and alternative splice variants that could be developmentally specific to the first larval stage. This work also demonstrates the successful and efficient application of a next generation sequencing methodology. RESULTS: We have generated over 30 million bases of novel expressed sequence tags from first larval stage worms utilizing high-throughput sequencing technology. We have shown that approximately 14% of the newly sequenced expressed sequence tags map completely or partially to genomic regions where there are no annotated genes or splice variants and therefore, imply that these are novel genetic structures. Expressed sequence tags, which map to intergenic (around 1000) and intronic regions (around 580), may represent novel transcribed regions, such as unannotated or unrecognized small protein-coding or non-protein-coding genes or splice variants. Expressed sequence tags, which map across intron-exon boundaries (around 300), indicate possible alternative splice sites, while expressed sequence tags, which map near the ends of known transcripts (around 600), suggest extension of the coding or untranslated regions. We have also discovered that intergenic and intronic expressed sequence tags, which are well conserved across different nematode species, are likely to represent non-coding RNAs. Lastly, we have incorporated available serial analysis of gene expression data generated from first larval stage worms, in order to predict novel transcripts that might be specifically or predominantly expressed in the first larval stage. CONCLUSION: We have demonstrated the use of a high-throughput sequencing methodology to efficiently produce a snap-shot of transcriptional activities occurring in the first larval stage of C. elegans development. Such application of this new sequencing technique allows for high-throughput, genome-wide experimental verification of known and novel transcripts. This study provides a more complete C. elegans transcriptome profile and, furthermore, gives insight into the evolutionary and biological complexity of this organism.

A physical map of the highly heterozygous Populus genome: integration with the genome sequence and genetic map and analysis of haplotype variation.

As part of a larger project to sequence the Populus genome and generate genomic resources for this emerging model tree, we constructed a physical map of the Populus genome, representing one of the few such maps of an undomesticated, highly heterozygous plant species. The physical map, consisting of 2802 contigs, was constructed from fingerprinted bacterial artificial chromosome (BAC) clones. The map represents approximately 9.4-fold coverage of the Populus genome, which has been estimated from the genome sequence assembly to be 485 +/- 10 Mb in size. BAC ends were sequenced to assist long-range assembly of whole-genome shotgun sequence scaffolds and to anchor the physical map to the genome sequence. Simple sequence repeat-based markers were derived from the end sequences and used to initiate integration of the BAC and genetic maps. A total of 2411 physical map contigs, representing 97% of all clones assigned to contigs, were aligned to the sequence assembly (JGI Populus trichocarpa, version 1.0). These alignments represent a total coverage of 384 Mb (79%) of the entire poplar sequence assembly and 295 Mb (96%) of linkage group sequence assemblies. A striking result of the physical map contig alignments to the sequence assembly was the co-localization of multiple contigs across numerous regions of the 19 linkage groups. Targeted sequencing of BAC clones and genetic analysis in a small number of representative regions showed that these co-aligning contigs represent distinct haplotypes in the heterozygous individual sequenced, and revealed the nature of these haplotype sequence differences.

The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse.

Rhodococcus sp. RHA1 (RHA1) is a potent polychlorinated biphenyl-degrading soil actinomycete that catabolizes a wide range of compounds and represents a genus of considerable industrial interest. RHA1 has one of the largest bacterial genomes sequenced to date, comprising 9,702,737 bp (67% G+C) arranged in a linear chromosome and three linear plasmids. A targeted insertion methodology was developed to determine the telomeric sequences. RHA1's 9,145 predicted protein-encoding genes are exceptionally rich in oxygenases (203) and ligases (192). Many of the oxygenases occur in the numerous pathways predicted to degrade aromatic compounds (30) or steroids (4). RHA1 also contains 24 nonribosomal peptide synthase genes, six of which exceed 25 kbp, and seven polyketide synthase genes, providing evidence that rhodococci harbor an extensive secondary metabolism. Among sequenced genomes, RHA1 is most similar to those of nocardial and mycobacterial strains. The genome contains few recent gene duplications. Moreover, three different analyses indicate that RHA1 has acquired fewer genes by recent horizontal transfer than most bacteria characterized to date and far fewer than Burkholderia xenovorans LB400, whose genome size and catabolic versatility rival those of RHA1. RHA1 and LB400 thus appear to demonstrate that ecologically similar bacteria can evolve large genomes by different means. Overall, RHA1 appears to have evolved to simultaneously catabolize a diverse range of plant-derived compounds in an O(2)-rich environment. In addition to establishing RHA1 as an important model for studying actinomycete physiology, this study provides critical insights that facilitate the exploitation of these industrially important microorganisms.

Mating factor linkage and genome evolution in basidiomycetous pathogens of cereals.

Sex in basidiomycete fungi is controlled by tetrapolar mating systems in which two unlinked gene complexes determine up to thousands of mating specificities, or by bipolar systems in which a single locus (MAT) specifies different sexes. The genus Ustilago contains bipolar (Ustilago hordei) and tetrapolar (Ustilago maydis) species and sexual development is associated with infection of cereal hosts. The U. hordei MAT-1 locus is unusually large (approximately 500 kb) and recombination is suppressed in this region. We mapped the genome of U. hordei and sequenced the MAT-1 region to allow a comparison with mating-type regions in U. maydis. Additionally the rDNA cluster in the U. hordei genome was identified and characterized. At MAT-1, we found 47 genes along with a striking accumulation of retrotransposons and repetitive DNA; the latter features were notably absent from the corresponding U. maydis regions. The tetrapolar mating system may be ancestral and differences in pathogenic life style and potential for inbreeding may have contributed to genome evolution.

A physical map of the genome of Atlantic salmon, Salmo salar.

A physical map of the Atlantic salmon (Salmo salar) genome was generated based on HindIII fingerprints of a publicly available BAC (bacterial artificial chromosome) library constructed from DNA isolated from a Norwegian male. Approximately 11.5 haploid genome equivalents (185,938 clones) were successfully fingerprinted. Contigs were first assembled via FPC using high-stringency (1e-16), and then end-to-end joins yielded 4354 contigs and 37,285 singletons. The accuracy of the contig assembly was verified by hybridization and PCR analysis using genetic markers. A subset of the BACs in the library contained few or no HindIII recognition sites in their insert DNA. BglI digestion fragment patterns of these BACs allowed us to identify three classes: (1) BACs containing histone genes, (2) BACs containing rDNA-repeating units, and (3) those that do not have BglI recognition sites. End-sequence analysis of selected BACs representing these three classes confirmed the identification of the first two classes and suggested that the third class contained highly repetitive DNA corresponding to tRNAs and related sequences.

A BAC-based physical map of the Drosophila buzzatii genome.

Large-insert genomic libraries facilitate cloning of large genomic regions, allow the construction of clone-based physical maps, and provide useful resources for sequencing entire genomes. Drosophila buzzatii is a representative species of the repleta group in the Drosophila subgenus, which is being widely used as a model in studies of genome evolution, ecological adaptation, and speciation. We constructed a Bacterial Artificial Chromosome (BAC) genomic library of D. buzzatii using the shuttle vector pTARBAC2.1. The library comprises 18,353 clones with an average insert size of 152 kb and an approximately 18x expected representation of the D. buzzatii euchromatic genome. We screened the entire library with six euchromatic gene probes and estimated the actual genome representation to be approximately 23x. In addition, we fingerprinted by restriction digestion and agarose gel electrophoresis a sample of 9555 clones, and assembled them using FingerPrint Contigs (FPC) software and manual editing into 345 contigs (mean of 26 clones per contig) and 670 singletons. Finally, we anchored 181 large contigs (containing 7788 clones) to the D. buzzatii salivary gland polytene chromosomes by in situ hybridization of 427 representative clones. The BAC library and a database with all the information regarding the high coverage BAC-based physical map described in this paper are available to the research community.

The genome of the basidiomycetous yeast and human pathogen Cryptococcus neoformans.

Cryptococcus neoformans is a basidiomycetous yeast ubiquitous in the environment, a model for fungal pathogenesis, and an opportunistic human pathogen of global importance. We have sequenced its approximately 20-megabase genome, which contains approximately 6500 intron-rich gene structures and encodes a transcriptome abundant in alternatively spliced and antisense messages. The genome is rich in transposons, many of which cluster at candidate centromeric regions. The presence of these transposons may drive karyotype instability and phenotypic variation. C. neoformans encodes unique genes that may contribute to its unusual virulence properties, and comparison of two phenotypically distinct strains reveals variation in gene content in addition to sequence polymorphisms between the genomes.

Functional characterization of a catabolic plasmid from polychlorinated- biphenyl-degrading Rhodococcus sp. strain RHA1.

Rhodococcus sp. strain RHA1, a potent polychlorinated-biphenyl (PCB)-degrading strain, contains three linear plasmids ranging in size from 330 to 1,100 kb. As part of a genome sequencing project, we report here the complete sequence and characterization of the smallest and least-well-characterized of the RHA1 plasmids, pRHL3. The plasmid is an actinomycete invertron, containing large terminal inverted repeats with a tightly associated protein and a predicted open reading frame (ORF) that is similar to that of a mycobacterial rep gene. The pRHL3 plasmid has 300 putative genes, almost 21% of which are predicted to have a catabolic function. Most of these are organized into three clusters. One of the catabolic clusters was predicted to include limonene degradation genes. Consistent with this prediction, RHA1 grew on limonene, carveol, or carvone as the sole carbon source. The plasmid carries three cytochrome P450-encoding (CYP) genes, a finding consistent with the high number of CYP genes found in other actinomycetes. Two of the CYP genes appear to belong to novel families; the third belongs to CYP family 116 but appears to belong to a novel class based on the predicted domain structure of its reductase. Analyses indicate that pRHL3 also contains four putative "genomic islands" (likely to have been acquired by horizontal transfer), insertion sequence elements, 19 transposase genes, and a duplication that spans two ORFs. One of the genomic islands appears to encode resistance to heavy metals. The plasmid does not appear to contain any housekeeping genes. However, each of the three catabolic clusters contains related genes that appear to be involved in glucose metabolism.