Addressing Futility: A Practical Approach.

Limiting or withdrawing nonbeneficial medical care is considered ethically responsible throughout most of critical care and medical ethics literature. Practically, however, setting limits to treatment is often challenging. We review the literature to identify best practices for using the definition of futility as an anchoring concept to aid the ethical practice of ICU clinicians. DATA SOURCES: Source data were obtained from a PubMed literature review. STUDY SELECTION: English language articles were chosen based on relevance to medical futility ethics, end-of-life care in the ICU, or communication and conflict mitigation strategies. DATA EXTRACTION: Independent evaluation of selected articles for recurrent content themes as relevant to our clinical case were compared among authors and based on consensus, quantitative and qualitative data from these sources were referenced directly. DATA SYNTHESIS: When life-sustaining treatment is unlikely to achieve a meaningful benefit such as symptom improvement, continued care may be discordant with the patient's goals. Institutional and cultural norms, unconscious biases, and difficulty with navigating conflicts all influence how un(comfortable) clinicians feel in setting limits to futile care. Defining futility in light of the patient's goals and values, focusing on outcomes rather than interventions, and being proactive in communication with families are the staples of medically meaningful critical care. Palliative measures should be framed affirmatively, and clinicians should be transparent about the limits of medicine. CONCLUSIONS: Clinicians have an ethical obligation not to provide futile care. To practice accordingly, we must clearly understand the nature and forms of futility. Armed with this understanding, our discussions with family and surrogates in the ICU should fundamentally comprise 1) eliciting the patient's values and goals, 2) communicating which interventions serve those values and goals and which do not, and 3) offering only those interventions whose likely outcomes are in line with said values and goals.

Critical Ethics: How to Balance Patient Autonomy With Fairness When Patients Refuse Coronavirus Disease 2019 Testing.

OBJECTIVES: A cornerstone of our healthcare system's response to the coronavirus disease 2019 pandemic is widespread testing to facilitate both isolation and early treatment. When patients refuse to undergo coronavirus disease testing, they compromise not only just their own health but also the health of those around them. The primary objective of our review is to identify the most ethical way a given healthcare system may respond to a patient's refusal to undergo coronavirus disease 2019 testing. DATA SOURCES: We apply a systematic approach to a true clinical case scenario to evaluate the ethical merits of four plausible responses to a patient's refusal to undergo coronavirus disease testing. Although our clinical case is anecdotal, it is representative of our experience at our University Tertiary Care Center. DATA EXTRACTION: Each plausible response in the case is rigorously analyzed by examining relevant stakeholders, facts, norms, and ethical weight both with respect to individuals' rights and to the interests of public health. We use the "So Far No Objections" method as the ethical approach of choice because it has been widely used in the Ethics Modules of the Surgical Council on Resident Education Curriculum of the American College of Surgeons. DATA SYNTHESIS: Two ethically viable options may be tailored to individual circumstances depending on the severity of the patient's condition. Although unstable patients must be assumed to be coronavirus disease positive and treated accordingly even in the absence of a test, stable patients who refuse testing may rightfully be asked to seek care elsewhere. CONCLUSIONS: Although patient autonomy is a fundamental principle of our society's medical ethic, during a pandemic we must, in the interest of vulnerable and critically ill patients, draw certain limits to obliging the preferences of noncritically ill patients with decisional capacity.

Ethics of Codes and Codes of Ethics: When Is It Ethical to Provide Cardiopulmonary Resuscitation During the COVID-19 Pandemic?

OBJECTIVE: Our study aims to provide a paradigm when it is ethical to perform cardiopulmonary resuscitation (CPR) on patients during the COVID-19 pandemic. SUMMARY BACKGROUND DATA: Hospitals around the nation are enacting systems to limit CPR in caring for COVID+ patients for a variety of legitimate reasons and based on concepts of medical futility and allocation of scarce resources. No ethical framework, however, has been proposed as a standard to guide care in this crucial matter. METHODS: Our analysis begins with definitions of ethically relevant terms. We then cycle an illustrative clinical vignette through the mathematically permissible possibilities to account for all conceivable scenarios. Scenarios with ethical tension are examined. RESULTS: Patients have the negative right to refuse care including CPR, but they do not have the positive right to demand it. Our detailed ethical analysis and recommendations support CPR if and only if 1) CPR is judged medically beneficial, and in line with the patient's and values and goals, 2) allocations or scarce resources follow a just and transparent triage system, and 3) providers are protected from contracting the disease. CONCLUSIONS: CPR is an intervention like any other, with attendant risks and benefits and with responsibility for the utilization of limited resources. Our ethical analysis advocates for a systematic approach to codes that respects the important ethical considerations in caring for the critically ill and facilitates patient-centered, evidence-based, and fair treatment to all.

Mechanistic insight into inhibition of two-component system signaling.

Two-component signal transduction systems (TCSs) are commonly used by bacteria to couple environmental stimuli to adaptive responses. Targeting the highly conserved kinase domain in these systems represents a promising strategy for the design of a broad-spectrum antibiotic; however, development of such compounds has been marred by an incomplete understanding of the conserved binding features within the active site that could be exploited in molecule design. Consequently, a large percentage of the available TCS inhibitors demonstrate poor target specificity and act via multiple mechanisms, with aggregation of the kinase being the most notable. In order to elucidate the mode of action of some of these compounds, molecular modeling was employed to dock a suite of molecules into the ATP-binding domain of several histidine kinases. This effort revealed a key structural feature of the domain that is likely interacting with several known inhibitors and is also highly conserved. Furthermore, generation of several simplified scaffolds derived from a reported inhibitor and characterization of these compounds using activity assays, protein aggregation studies and saturation transfer differential (STD) NMR suggests that targeting of this protein feature may provide a basis for the design of ATP-competitive compounds.

Diverse and tissue-enriched small RNAs in the plant pathogenic fungus, Magnaporthe oryzae.

BACKGROUND: Emerging knowledge of the impact of small RNAs as important cellular regulators has prompted an explosion of small transcriptome sequencing projects. Although significant progress has been made towards small RNA discovery and biogenesis in higher eukaryotes and other model organisms, knowledge in simple eukaryotes such as filamentous fungi remains limited. RESULTS: Here, we used 454 pyrosequencing to present a detailed analysis of the small RNA transcriptome (~ 15 - 40 nucleotides in length) from mycelia and appressoria tissues of the rice blast fungal pathogen, Magnaporthe oryzae. Small RNAs mapped to numerous nuclear and mitochondrial genomic features including repetitive elements, tRNA loci, rRNAs, protein coding genes, snRNAs and intergenic regions. For most elements, small RNAs mapped primarily to the sense strand with the exception of repetitive elements to which small RNAs mapped in the sense and antisense orientation in near equal proportions. Inspection of the small RNAs revealed a preference for U and suppression of C at position 1, particularly for antisense mapping small RNAs. In the mycelia library, small RNAs of the size 18 - 23 nt were enriched for intergenic regions and repetitive elements. Small RNAs mapping to LTR retrotransposons were classified as LTR retrotransposon-siRNAs (LTR-siRNAs). Conversely, the appressoria library had a greater proportion of 28 - 35 nt small RNAs mapping to tRNA loci, and were classified as tRNA-derived RNA fragments (tRFs). LTR-siRNAs and tRFs were independently validated by 3' RACE PCR and northern blots, respectively. CONCLUSIONS: Our findings suggest M. oryzae small RNAs differentially accumulate in vegetative and specialized-infection tissues and may play an active role in genome integrity and regulating growth and development.

Genome-wide characterization of methylguanosine-capped and polyadenylated small RNAs in the rice blast fungus Magnaporthe oryzae.

Small RNAs are well described in higher eukaryotes such as mammals and plants; however, knowledge in simple eukaryotes such as filamentous fungi is limited. In this study, we discovered and characterized methylguanosine-capped and polyadenylated small RNAs (CPA-sRNAs) by using differential RNA selection, full-length cDNA cloning and 454 transcriptome sequencing of the rice blast fungus Magnaporthe oryzae. This fungus causes blast, a devastating disease on rice, the principle food staple for over half the world's population. CPA-sRNAs mapped primarily to the transcription initiation and termination sites of protein-coding genes and were positively correlated with gene expression, particularly for highly expressed genes including those encoding ribosomal proteins. Numerous CPA-sRNAs also mapped to rRNAs, tRNAs, snRNAs, transposable elements and intergenic regions. Many other 454 sequence reads could not be mapped to the genome; however, inspection revealed evidence for non-template additions and chimeric sequences. CPA-sRNAs were independently confirmed using a high affinity variant of eIF-4E to capture 5'-methylguanosine-capped RNA followed by 3'-RACE sequencing. These results expand the repertoire of small RNAs in filamentous fungi.

Induction of an IL7-R(+)c-Kit(hi) myelolymphoid progenitor critically dependent on IFN-gamma signaling during acute malaria.

Although the relationship between hematopoietic stem cells and progenitor populations has been investigated extensively under steady-state conditions, the dynamic response of the hematopoietic compartment during acute infection is largely unknown. Here we show that after infection of mice with Plasmodium chabaudi, a c-Kit(hi) progenitor subset positive for interleukin 7 receptor-alpha (IL-7Ralpha) emerged that had both lymphoid and myeloid potential in vitro. After being transferred into uninfected alymphoid or malaria-infected hosts, IL-7Ralpha(+)c-Kit(hi) progenitors generated mainly myeloid cells that contributed to the clearance of infected erythrocytes in infected hosts. The generation of these infection-induced progenitors was critically dependent on interferon-gamma (IFN-gamma) signaling in hematopoietic progenitors. Thus, IFN-gamma is a key modulator of hematopoiesis and innate and adaptive immunity during acute malaria infection.

Gene Ontology annotation of the rice blast fungus, Magnaporthe oryzae.

BACKGROUND: Magnaporthe oryzae, the causal agent of blast disease of rice, is the most destructive disease of rice worldwide. The genome of this fungal pathogen has been sequenced and an automated annotation has recently been updated to Version 6 http://www.broad.mit.edu/annotation/genome/magnaporthe_grisea/MultiDownloads.html. However, a comprehensive manual curation remains to be performed. Gene Ontology (GO) annotation is a valuable means of assigning functional information using standardized vocabulary. We report an overview of the GO annotation for Version 5 of M. oryzae genome assembly. METHODS: A similarity-based (i.e., computational) GO annotation with manual review was conducted, which was then integrated with a literature-based GO annotation with computational assistance. For similarity-based GO annotation a stringent reciprocal best hits method was used to identify similarity between predicted proteins of M. oryzae and GO proteins from multiple organisms with published associations to GO terms. Significant alignment pairs were manually reviewed. Functional assignments were further cross-validated with manually reviewed data, conserved domains, or data determined by wet lab experiments. Additionally, biological appropriateness of the functional assignments was manually checked. RESULTS: In total, 6,286 proteins received GO term assignment via the homology-based annotation, including 2,870 hypothetical proteins. Literature-based experimental evidence, such as microarray, MPSS, T-DNA insertion mutation, or gene knockout mutation, resulted in 2,810 proteins being annotated with GO terms. Of these, 1,673 proteins were annotated with new terms developed for Plant-Associated Microbe Gene Ontology (PAMGO). In addition, 67 experiment-determined secreted proteins were annotated with PAMGO terms. Integration of the two data sets resulted in 7,412 proteins (57%) being annotated with 1,957 distinct and specific GO terms. Unannotated proteins were assigned to the 3 root terms. The Version 5 GO annotation is publically queryable via the GO site http://amigo.geneontology.org/cgi-bin/amigo/go.cgi. Additionally, the genome of M. oryzae is constantly being refined and updated as new information is incorporated. For the latest GO annotation of Version 6 genome, please visit our website http://scotland.fgl.ncsu.edu/smeng/GoAnnotationMagnaporthegrisea.html. The preliminary GO annotation of Version 6 genome is placed at a local MySql database that is publically queryable via a user-friendly interface Adhoc Query System. CONCLUSION: Our analysis provides comprehensive and robust GO annotations of the M. oryzae genome assemblies that will be solid foundations for further functional interrogation of M. oryzae.

Transcriptome analysis reveals new insight into appressorium formation and function in the rice blast fungus Magnaporthe oryzae.

BACKGROUND: Rice blast disease is caused by the filamentous Ascomycetous fungus Magnaporthe oryzae and results in significant annual rice yield losses worldwide. Infection by this and many other fungal plant pathogens requires the development of a specialized infection cell called an appressorium. The molecular processes regulating appressorium formation are incompletely understood. RESULTS: We analyzed genome-wide gene expression changes during spore germination and appressorium formation on a hydrophobic surface compared to induction by cAMP. During spore germination, 2,154 (approximately 21%) genes showed differential expression, with the majority being up-regulated. During appressorium formation, 357 genes were differentially expressed in response to both stimuli. These genes, which we refer to as appressorium consensus genes, were functionally grouped into Gene Ontology categories. Overall, we found a significant decrease in expression of genes involved in protein synthesis. Conversely, expression of genes associated with protein and amino acid degradation, lipid metabolism, secondary metabolism and cellular transportation exhibited a dramatic increase. We functionally characterized several differentially regulated genes, including a subtilisin protease (SPM1) and a NAD specific glutamate dehydrogenase (Mgd1), by targeted gene disruption. These studies revealed hitherto unknown findings that protein degradation and amino acid metabolism are essential for appressorium formation and subsequent infection. CONCLUSION: We present the first comprehensive genome-wide transcript profile study and functional analysis of infection structure formation by a fungal plant pathogen. Our data provide novel insight into the underlying molecular mechanisms that will directly benefit efforts to identify fungal pathogenicity factors and aid the development of new disease management strategies.

Altered patterns of gene duplication and differential gene gain and loss in fungal pathogens.

BACKGROUND: Duplication, followed by fixation or random loss of novel genes, contributes to genome evolution. Particular outcomes of duplication events are possibly associated with pathogenic life histories in fungi. To date, differential gene gain and loss have not been studied at genomic scales in fungal pathogens, despite this phenomenon's known importance in virulence in bacteria and viruses. RESULTS: To determine if patterns of gene duplication differed between pathogens and non-pathogens, we identified gene families across nine euascomycete and two basidiomycete species. Gene family size distributions were fit to power laws to compare gene duplication trends in pathogens versus non-pathogens. Fungal phytopathogens showed globally altered patterns of gene duplication, as indicated by differences in gene family size distribution. We also identified sixteen examples of gene family expansion and five instances of gene family contraction in pathogenic lineages. Expanded gene families included those predicted to be important in melanin biosynthesis, host cell wall degradation and transport functions. Contracted families included those encoding genes involved in toxin production, genes with oxidoreductase activity, as well as subunits of the vacuolar ATPase complex. Surveys of the functional distribution of gene duplicates indicated that pathogens show enrichment for gene duplicates associated with receptor and hydrolase activities, while euascomycete pathogens appeared to have not only these differences, but also significantly more duplicates associated with regulatory and carbohydrate binding functions. CONCLUSION: Differences in the overall levels of gene duplication in phytopathogenic species versus non-pathogenic relatives implicate gene inventory flux as an important virulence-associated process in fungi. We hypothesize that the observed patterns of gene duplicate enrichment, gene family expansion and contraction reflect adaptation within pathogenic life histories. These adaptations were likely shaped by ancient, as well as contemporary, intimate associations with monocot hosts.

GT-Miner: a graph-theoretic data miner, viewer, and model processor.

UNLABELLED: Inexpensive computational power combined with high-throughput experimental platforms has created a wealth of biological information requiring analytical tools and techniques for interpretation. Graph-theoretic concepts and tools have provided an important foundation for information visualization, integration, and analysis of datasets, but they have often been relegated to background analysis tasks. GT-Miner is designed for visual data analysis and mining operations, interacts with other software, including databases, and works with diverse data types. It facilitates a discovery-oriented approach to data mining wherein exploration of alterations of the data and variations of the visualization is encouraged. The user is presented with a basic iterative process, consisting of loading, visualizing, transforming, and then storing the resultant information. Complex analyses are built-up through repeated iterations and user interactions. The iterative process is optimized by automatic layout following transformations and by maintaining a current selection set of interest for elements modified by the transformations. Multiple visualizations are supported including hierarchical, spring, and force-directed self-organizing layouts. Graphs can be transformed with an extensible set of algorithms or manually with an integral visual editor. GT-Miner is intended to allow easier access to visual data mining for the non-expert. AVAILABILITY: The GT-Miner program and supplemental materials, including example uses and a user guide, are freely available from http://www.cifr.ncsu.edu/bioinformatics/downloads/

Anti-HIV activity of steric block oligonucleotides.

The unabated increase in spread of HIV infection worldwide has redoubled efforts to discover novel antiviral and virucidal agents that might be starting points for clinical development. Oligonucleotides and their analogs targeted to form complementary duplexes with highly conserved regions of the HIV RNA have shown significant antiviral activity, but to date clinical studies have been dominated by RNase H-inducing oligonucleotide analog phosphorothioates (GEM 91 and 92) that have specificity and efficacy limitations. However, they have proven the principle that oligonucleotides can be safe anti-HIV drugs. Newer oligonucleotide analogs are now available, which act as strong steric block agents of HIV RNA function. We describe our ongoing studies targeting the HIV-1 trans-activation responsive region (TAR) and the viral packaging signal (psi) with steric block oligonucleotides of varying chemistry and demonstrate their great potential for steric blocking of viral protein interactions in vitro and in cells and describe the first antiviral studies. Peptide nucleic acids (PNA) disulfide linked to cell-penetrating peptides (CPP) have been found to have particular promise for the lipid-free direct delivery into cultured cells and are excellent candidates for their development as antiviral and virucidal agents.

Insights into the immunopathogenesis of malaria using mouse models.

Malaria kills approximately 1-2 million people every year, mostly in sub-Saharan Africa and in Asia. These deaths are at the most severe end of a scale of pathologies affecting approximately 500 million people per year. Much of the pathogenesis of malaria is caused by inappropriate or excessive immune responses mounted by the body to eliminate malaria parasites. In this review, we examine the evidence that immunopathology is responsible for malaria disease in the context of what we have learnt from animal models of malaria. In particular, we look in detail at the processes involved in endothelial cell damage leading to syndromes such as cerebral malaria, as well as generalised systemic manifestations such as anaemia, cachexia and problems with thermoregulation of the body. We also consider malaria in light of the variation of the severity of disease observed among people, and discuss the contribution from animal models to our understanding of this variation. Finally, we discuss some of the implications of immunopathology, and of host and parasite genetic variation, for the design and implementation of anti-malarial vaccines.