Profiling COVID-19 Genetic Research: A Data-Driven Study Utilizing Intelligent Bibliometrics.

The COVID-19 pandemic constitutes an ongoing worldwide threat to human society and has caused massive impacts on global public health, the economy and the political landscape. The key to gaining control of the disease lies in understanding the genetics of SARS-CoV-2 and the disease spectrum that follows infection. This study leverages traditional and intelligent bibliometric methods to conduct a multi-dimensional analysis on 5,632 COVID-19 genetic research papers, revealing that 1) the key players include research institutions from the United States, China, Britain and Canada; 2) research topics predominantly focus on virus infection mechanisms, virus testing, gene expression related to the immune reactions and patient clinical manifestation; 3) studies originated from the comparison of SARS-CoV-2 to previous human coronaviruses, following which research directions diverge into the analysis of virus molecular structure and genetics, the human immune response, vaccine development and gene expression related to immune responses; and 4) genes that are frequently highlighted include ACE2, IL6, TMPRSS2, and TNF. Emerging genes to the COVID-19 consist of FURIN, CXCL10, OAS1, OAS2, OAS3, and ISG15. This study demonstrates that our suite of novel bibliometric tools could help biomedical researchers follow this rapidly growing field and provide substantial evidence for policymakers' decision-making on science policy and public health administration.

DNA extraction from placental, fetal and neonatal tissue at autopsy: what organ to sample for DNA in the genomic era?

Incorporation of genome and exome sequencing into fetal and neonatal autopsy investigations has been shown to improve diagnostic yield. This requires deoxyribonucleic acid (DNA) to be extracted from either the placenta or autopsy tissue for molecular testing. However, the sources and quality of DNA obtained are highly variable and there are no adequate published data on what tissue is most ideal to sample for DNA extraction in this setting. Here we compare the quality of DNA extracted from sampling the placenta and various solid organs at fetal and neonatal autopsy, thereby determining the optimal tissue from which to source DNA for ancillary testing as part of the modern perinatal autopsy. A total of 898 tissue samples were obtained at autopsy from 176 fetuses (gestational ages 17-40 weeks) and 44 neonates (age range 0-28 days) at our tertiary institution. Fetal tissue was processed using the QIAsymphony DSP DNA Mini kit and placental tissue was extracted using the New iGENatal Kit. DNA concentration was quantified using the Qubit dsDNA BR Assay Kit. DNA integrity, as stratified by gel electrophoresis was classified as high (≥5 kb) or low quality (<5 kb). Genome sequencing was performed on the extracted DNA, together with respective parental DNA from blood samples, and confirmed absence of maternal contamination in all cases. Analyses used logistic mixed models to test for associations between tissue types, intrauterine retention times, delivery to autopsy and death to autopsy intervals with DNA quality. In the fetal cohort, the placenta had the highest proportion of high quality DNA samples (93.1%), and liver had the lowest proportion (35.3%). Among the neonates, all tissue samples with the exception of liver had over 88% high DNA quality with the placenta also yielding the highest quality (100%). There was statistically significant deterioration in DNA quality with prolonged time interval between demise and autopsy (≥5 days). In the 726 fetal samples, the odds of obtaining higher quality DNA from the placenta, thymus, and spleen were 70.4 [95% confidence interval (CI) 29.2-169.6], 3.6 (95% CI 2.0-6.6) and 3.3 (95% CI 1.8-6.1) times, respectively, more likely than samples from the liver (p values <0.001). DNA yield from other fetal solid organs investigated was not significantly superior to that from the liver. This study shows that, when available, refrigerated unfixed placenta is the most suitable source of high quality DNA during perinatal investigations. Of the solid fetal organs sampled at autopsy, lymphocyte-rich, lytic enzymes-poor organs such as thymus and spleen were significantly more likely to yield good quality DNA than the liver.

Functional Analysis of Genetic Variation in the SECIS Element of Thyroid Hormone Activating Type 2 Deiodinase.

CONTEXT: Thyroid hormone is important for normal brain development. The type 2 deiodinase (D2) controls thyroid hormone action in the brain by activating T4 to T3. The enzymatic activity of D2 depends on the incorporation of selenocysteine for which the selenocysteine-insertion sequence (SECIS) element located in the 3' untranslated region is indispensable. We hypothesized that mutations in the SECIS element could affect D2 function, resulting in a neurocognitive phenotype. OBJECTIVE: To identify mutations in the SECIS element of DIO2 in patients with intellectual disability and to test their functional consequences. DESIGN, SETTING, AND PATIENTS: The SECIS element of DIO2 was sequenced in 387 patients with unexplained intellectual disability using a predefined pattern of thyroid function tests. SECIS element read-through in wild-type or mutant D2 was quantified by a luciferase reporter system in transfected cells. Functional consequences were assessed by quantifying D2 activity in cell lysate or intact cell metabolism studies. RESULTS: Sequence analysis revealed 2 heterozygous mutations: c.5703C>T and c.5730A>T, which were also present in the unaffected family members. The functional evaluation showed that both mutations did not affect D2 enzyme activity in cell lysates or intact cells, although the 5730A>T mutation decreased SECIS element read-through by 75%. In the patient harboring the c.5730A>T variant, whole genome sequencing revealed a pathogenic deletion of the STXBP1 gene. CONCLUSIONS: We report on two families with mutations in the SECIS element of D2. Although functional analysis showed that nucleotide 5730 is important for normal SECIS element read-through, the two variants did not segregate with a distinct phenotype.

L1 Retrotransposon Heterogeneity in Ovarian Tumor Cell Evolution.

LINE-1 (L1) retrotransposons are a source of insertional mutagenesis in tumor cells. However, the clinical significance of L1 mobilization during tumorigenesis remains unclear. Here, we applied retrotransposon capture sequencing (RC-seq) to multiple single-cell clones isolated from five ovarian cancer cell lines and HeLa cells and detected endogenous L1 retrotransposition in vitro. We then applied RC-seq to ovarian tumor and matched blood samples from 19 patients and identified 88 tumor-specific L1 insertions. In one tumor, an intronic de novo L1 insertion supplied a novel cis-enhancer to the putative chemoresistance gene STC1. Notably, the tumor subclone carrying the STC1 L1 mutation increased in prevalence after chemotherapy, further increasing STC1 expression. We also identified hypomethylated donor L1s responsible for new L1 insertions in tumors and cultivated cancer cells. These congruent in vitro and in vivo results highlight L1 insertional mutagenesis as a common component of ovarian tumorigenesis and cancer genome heterogeneity.

A point mutation in the pre-ZRS disrupts sonic hedgehog expression in the limb bud and results in triphalangeal thumb-polysyndactyly syndrome.

PURPOSE: The zone of polarizing activity regulatory sequence (ZRS) is an enhancer that regulates sonic hedgehog during embryonic limb development. Recently, mutations in a noncoding evolutionary conserved sequence 500 bp upstream of the ZRS, termed the pre-ZRS (pZRS), have been associated with polydactyly in dogs and humans. Here, we report the first case of triphalangeal thumb-polysyndactyly syndrome (TPT-PS) to be associated with mutations in this region and show via mouse enhancer assays how this mutation leads to ectopic expression throughout the developing limb bud. METHODS: We used linkage analysis, whole-exome sequencing, Sanger sequencing, fluorescence in situ hybridization, multiplex ligation-dependent probe amplification, single-nucleotide polymorphism array, and a mouse transgenic enhancer assay. RESULTS: Ten members of a TPT-PS family were included in this study. The mutation was linked to chromosome 7q36 (LOD score 3.0). No aberrations in the ZRS could be identified. A point mutation in the pZRS (chr7:156585476G>C; GRCh37/hg19) was detected in all affected family members. Functional characterization using a mouse transgenic enhancer essay showed extended ectopic expression dispersed throughout the entire limb bud (E11.5). CONCLUSION: Our work describes the first mutation in the pZRS to be associated with TPT-PS and provides functional evidence that this mutation leads to ectopic expression of this enhancer within the developing limb.

Application of Whole Genome Sequencing Technology in the Investigation of Genetic Causes of Fetal, Perinatal, and Early Infant Death.

Death in the fetal, perinatal, and early infant age-group has a multitude of causes, a proportion of which is presumed to be genetic. Defining a specific genetic aberration leading to the death is problematic at this young age, due to limited phenotype-genotype correlation inherent in the underdeveloped phenotype, the inability to assess certain phenotypic traits after death, and the problems of dealing with rare disorders. In this study, our aim was to increase the yield of identification of a defined genetic cause of an early death. Therefore, we employed whole genome sequencing and bioinformatic filtering techniques as a comprehensive, unbiased genetic investigation into 16 fetal, perinatal, and early infant deaths, which had undergone a full autopsy. A likely genetic cause was identified in two cases (in genes; COL2A1 and RYR1) and a speculative genetic cause in a further six cases (in genes: ARHGAP35, BBS7, CASZ1, CRIM1, DHCR7, HADHB, HAPLN3, HSPG2, MYO18B, and SRGAP2). This investigation indicates that whole genome sequencing is a significantly enabling technology when determining genetic causes of early death.

Isolated Ventricular Noncompaction Cardiomyopathy Presenting as Fetal Hydrops at 24 Weeks Gestation.

Ventricular noncompaction cardiomyopathy is a rare form of congenital cardiomyopathy with increasing evidence of genetic etiology, especially when presenting in childhood. Fetal presentation is rare. We describe a case of fetal hydrops, presenting at 24 weeks gestation and leading to intrapartum death at 26 weeks gestation. Autopsy examination revealed characteristic features of left ventricular noncompaction. A genetic analysis identified a constellation of variants of unknown significance in MYH6, TNNC1, and MYBPC3, genes known to be important in sarcomeric function. Additionally, the variant in MYBPC3 was homozygous. While this case did not demonstrate a conventional single-gene mutation as the cause of the ventricular noncompaction, a broader genomic investigation revealed several variants in sarcomeric genes which may act synergistically to impact cardiac function.

Macrolide Treatment Inhibits Pseudomonas aeruginosa Quorum Sensing in Non-Cystic Fibrosis Bronchiectasis. An Analysis from the Bronchiectasis and Low-Dose Erythromycin Study Trial.

RATIONALE: The mechanism by which low-dose macrolide therapy reduces exacerbations in non-cystic fibrosis bronchiectasis is not known. Pseudomonas aeruginosa quorum sensing controls the expression of a range of pathogenicity traits and is inhibited by macrolide in vitro. Quorum sensing inhibition renders P. aeruginosa less pathogenic, potentially reducing its contribution to airway damage. OBJECTIVES: The aim of this study was to determine whether long-term low-dose erythromycin inhibits P. aeruginosa quorum sensing within the airways of patients with non-cystic fibrosis bronchiectasis. METHODS: Analysis was performed on induced sputum from P. aeruginosa-positive subjects at recruitment to the BLESS (Bronchiectasis and Low-Dose Erythromycin Study) trial and after 48 weeks of treatment with erythromycin or placebo. To avoid changes in gene expression during culture, bacterial mRNA was extracted directly from sputum, and the relative expression of functionally critical quorum sensing genes was determined by quantitative polymerase chain reaction. MEASUREMENTS AND MAIN RESULTS: In keeping with the BLESS study, a significant reduction in total exacerbations was seen in this subgroup (placebo: 6, [interquartile range (IQR), 4-8]; erythromycin: 3, [IQR, 3-4]; P = 0.008; Mann-Whitney test). Erythromycin therapy did not change P. aeruginosa bacterial load determined by polymerase chain reaction. A significant reduction was observed in the expression of the quorum sensing genes, lasR (erythromycin: fold change, 0.065 [IQR, 0.01-0.85], n = 11; placebo: fold change, 1.000 [IQR, 0.05-3.05]; P = 0.047, Mann-Whitney U test) and pqsA (erythromycin: fold change, 0.07 [IQR, 0.02-0.25]; placebo: fold change, 1.000 [IQR, 0.21-4.31], P = 0.017, Mann-Whitney U test), after 48 weeks of erythromycin, compared with placebo. CONCLUSIONS: We demonstrate inhibition of P. aeruginosa quorum sensing within the airways of patients with non-cystic fibrosis bronchiectasis receiving long-term, low-dose erythromycin, without a reduction in bacterial load, representing a potential mechanism of therapeutic impact beyond a classical antimicrobial or antiinflammatory pathway.

Ovarian Microcystic Stromal Tumor: A Rare Clinical Manifestation of Familial Adenomatous Polyposis.

Microcystic stromal tumor (MST) is a rare tumor of presumed sex-cord stromal differentiation. We present a case of MST arising within a patient with constitutional 5q deletion syndrome, whose deletion encompassed the APC gene. Genomic analysis of the MST revealed a point mutation in the remaining APC allele, predicted to result in abnormal splicing of Exon 7. Subsequent clinical investigation revealed multiple gastrointestinal polyps qualifying for a diagnosis of familial adenomatous polyposis. This case emphasizes the importance of an aberrant Wnt/ß-catenin pathway in the development of MST and adds credence to the inclusion of MST as a rare phenotype of familial adenomatous polyposis. In a search for additional genetic aberrations which may contribute to the development of this rare tumor, genomic analysis revealed a frameshift mutation in FANCD2, a protein which plays a key role in DNA repair. This protein is expressed in human ovarian stromal cells and FANCD2-knockout mice are known to develop sex cord-stromal tumors, factors which further support a possible role of aberrant FANCD2 in the development of MST.

A Molecular Host Response Assay to Discriminate Between Sepsis and Infection-Negative Systemic Inflammation in Critically Ill Patients: Discovery and Validation in Independent Cohorts.

BACKGROUND: Systemic inflammation is a whole body reaction having an infection-positive (i.e., sepsis) or infection-negative origin. It is important to distinguish between these two etiologies early and accurately because this has significant therapeutic implications for critically ill patients. We hypothesized that a molecular classifier based on peripheral blood RNAs could be discovered that would (1) determine which patients with systemic inflammation had sepsis, (2) be robust across independent patient cohorts, (3) be insensitive to disease severity, and (4) provide diagnostic utility. The goal of this study was to identify and validate such a molecular classifier. METHODS AND FINDINGS: We conducted an observational, non-interventional study of adult patients recruited from tertiary intensive care units (ICUs). Biomarker discovery utilized an Australian cohort (n = 105) consisting of 74 cases (sepsis patients) and 31 controls (post-surgical patients with infection-negative systemic inflammation) recruited at five tertiary care settings in Brisbane, Australia, from June 3, 2008, to December 22, 2011. A four-gene classifier combining CEACAM4, LAMP1, PLA2G7, and PLAC8 RNA biomarkers was identified. This classifier, designated SeptiCyte Lab, was validated using reverse transcription quantitative PCR and receiver operating characteristic (ROC) curve analysis in five cohorts (n = 345) from the Netherlands. Patients for validation were selected from the Molecular Diagnosis and Risk Stratification of Sepsis study (ClinicalTrials.gov, NCT01905033), which recruited ICU patients from the Academic Medical Center in Amsterdam and the University Medical Center Utrecht. Patients recruited from November 30, 2012, to August 5, 2013, were eligible for inclusion in the present study. Validation cohort 1 (n = 59) consisted entirely of unambiguous cases and controls; SeptiCyte Lab gave an area under curve (AUC) of 0.95 (95% CI 0.91-1.00) in this cohort. ROC curve analysis of an independent, more heterogeneous group of patients (validation cohorts 2-5; 249 patients after excluding 37 patients with an infection likelihood of "possible") gave an AUC of 0.89 (95% CI 0.85-0.93). Disease severity, as measured by Sequential Organ Failure Assessment (SOFA) score or Acute Physiology and Chronic Health Evaluation (APACHE) IV score, was not a significant confounding variable. The diagnostic utility of SeptiCyte Lab was evaluated by comparison to various clinical and laboratory parameters available to a clinician within 24 h of ICU admission. SeptiCyte Lab was significantly better at differentiating cases from controls than all tested parameters, both singly and in various logistic combinations, and more than halved the diagnostic error rate compared to procalcitonin in all tested cohorts and cohort combinations. Limitations of this study relate to (1) cohort compositions that do not perfectly reflect the composition of the intended use population, (2) potential biases that could be introduced as a result of the current lack of a gold standard for diagnosing sepsis, and (3) lack of a complete, unbiased comparison to C-reactive protein. CONCLUSIONS: SeptiCyte Lab is a rapid molecular assay that may be clinically useful in managing ICU patients with systemic inflammation. Further study in population-based cohorts is needed to validate this assay for clinical use.

Production and packaging of a biological arsenal: evolution of centipede venoms under morphological constraint.

Venom represents one of the most extreme manifestations of a chemical arms race. Venoms are complex biochemical arsenals, often containing hundreds to thousands of unique protein toxins. Despite their utility for prey capture, venoms are energetically expensive commodities, and consequently it is hypothesized that venom complexity is inversely related to the capacity of a venomous animal to physically subdue prey. Centipedes, one of the oldest yet least-studied venomous lineages, appear to defy this rule. Although scutigeromorph centipedes produce less complex venom than those secreted by scolopendrid centipedes, they appear to rely heavily on venom for prey capture. We show that the venom glands are large and well developed in both scutigerid and scolopendrid species, but that scutigerid forcipules lack the adaptations that allow scolopendrids to inflict physical damage on prey and predators. Moreover, we reveal that scolopendrid venom glands have evolved to accommodate a much larger number of secretory cells and, by using imaging mass spectrometry, we demonstrate that toxin production is heterogeneous across these secretory units. We propose that the differences in venom complexity between centipede orders are largely a result of morphological restrictions of the venom gland, and consequently there is a strong correlation between the morphological and biochemical complexity of this unique venom system. The current data add to the growing body of evidence that toxins are not expressed in a spatially homogenous manner within venom glands, and they suggest that the link between ecology and toxin evolution is more complex than previously thought.

Multifunctional warheads: diversification of the toxin arsenal of centipedes via novel multidomain transcripts.

Arthropod toxins are almost invariably encoded by transcripts encoding prepropeptides that are posttranslationally processed to yield a single mature toxin. In striking contrast to this paradigm, we used a complementary transcriptomic, proteomic and MALDI-imaging approach to identify four classes of multidomain centipede-toxin transcripts that each encodes multiple mature toxins. These multifunctional warheads comprise either: (1) repeats of linear peptides; (2) linear peptides preceding cysteine-rich peptides; (3) cysteine-rich peptides preceding linear peptides; or (4) repeats of linear peptides preceding cysteine-rich peptides. MALDI imaging of centipede venom glands revealed that these peptides are posttranslationally liberated from the original gene product in the venom gland and not by proteases following venom secretion. These multidomain transcripts exhibit a remarkable conservation of coding sequences, in striking contrast to monodomain toxin transcripts from related centipede species, and we demonstrate that they represent a rare class of predatory toxins that have evolved under strong negative selection. We hypothesize that the peptide toxins liberated from multidomain precursors might have synergistic modes of action, thereby allowing negative selection to dominate as the toxins encoded by the same transcript become increasingly interdependent. BIOLOGICAL SIGNIFICANCE: These results have direct implications for understanding the evolution of centipede venoms, and highlight the importance of taking a multidisciplinary approach for the investigation of novel venoms. The potential synergistic actions of the mature peptides are also of relevance to the growing biodiscovery efforts aimed at centipede venom. We also demonstrate the application of MALDI imaging in providing a greater understanding of toxin production in venom glands. This is the first MALDI imaging data of any venom gland.

Evolution of separate predation- and defence-evoked venoms in carnivorous cone snails.

Venomous animals are thought to inject the same combination of toxins for both predation and defence, presumably exploiting conserved target pharmacology across prey and predators. Remarkably, cone snails can rapidly switch between distinct venoms in response to predatory or defensive stimuli. Here, we show that the defence-evoked venom of Conus geographus contains high levels of paralytic toxins that potently block neuromuscular receptors, consistent with its lethal effects on humans. In contrast, C. geographus predation-evoked venom contains prey-specific toxins mostly inactive at human targets. Predation- and defence-evoked venoms originate from the distal and proximal regions of the venom duct, respectively, explaining how different stimuli can generate two distinct venoms. A specialized defensive envenomation strategy is widely evolved across worm, mollusk and fish-hunting cone snails. We propose that defensive toxins, originally evolved in ancestral worm-hunting cone snails to protect against cephalopod and fish predation, have been repurposed in predatory venoms to facilitate diversification to fish and mollusk diets.

Inosine triphosphate pyrophosphohydrolase (ITPA) polymorphic sequence variants in adult hematological malignancy patients and possible association with mitochondrial DNA defects.

BACKGROUND: Inosine triphosphate pyrophosphohydrolase (ITPase) is a 'house-cleaning' enzyme that degrades non-canonical ('rogue') nucleotides. Complete deficiency is fatal in knockout mice, but a mutant polymorphism resulting in low enzyme activity with an accumulation of ITP and other non-canonical nucleotides, appears benign in humans. We hypothesised that reduced ITPase activity may cause acquired mitochondrial DNA (mtDNA) defects. Furthermore, we investigated whether accumulating mtDNA defects may then be a risk factor for cell transformation, in adult haematological malignancy (AHM). METHODS: DNA was extracted from peripheral blood and bone marrow samples. Microarray-based sequencing of mtDNA was performed on 13 AHM patients confirmed as carrying the ITPA 94C>A mutation causing low ITPase activity, and 4 AHM patients with wildtype ITPA. The frequencies of ITPA 94C>A and IVS2+21A>C polymorphisms were studied from 85 available AHM patients. RESULTS: ITPA 94C>A was associated with a significant increase in total heteroplasmic/homoplasmic mtDNA mutations (p<0.009) compared with wildtype ITPA, following exclusion of haplogroup variants. This suggested that low ITPase activity may induce mitochondrial abnormalities. Compared to the normal population, frequencies for the 94C>A and IVS2+21A>C mutant alleles among the AHM patients were higher for myelodyplastic syndrome (MDS) - but below significance; were approximately equivalent for chronic lymphoblastic leukemia; and were lower for acute myeloid leukemia. CONCLUSIONS: This study invokes a new paradigm for the evolution of MDS, where nucleotide imbalances produced by defects in 'house-cleaning' genes may induce mitochondrial dysfunction, compromising cell integrity. It supports recent studies which point towards an important role for ITPase in cellular surveillance of rogue nucleotides.

Distinct roles for Toll and autophagy pathways in double-stranded RNA toxicity in a Drosophila model of expanded repeat neurodegenerative diseases.

Dominantly inherited expanded repeat neurodegenerative diseases are caused by the expansion of variable copy number tandem repeat sequences in otherwise unrelated genes. Some repeats encode polyglutamine that is thought to be toxic; however, other repeats do not encode polyglutamine indicating either multiple pathogenic pathways or an alternative common toxic agent. As these diseases share numerous clinical features and expanded repeat RNA is a common intermediary, RNA-based pathogenesis has been proposed, based on its toxicity in animal models. In Drosophila, double-stranded (rCAG.rCUG∼100) RNA toxicity is Dicer dependent and generates single-stranded (rCAG)7, an entity also detected in affected Huntington's Disease (HD) brains. We demonstrate that Drosophila rCAG.rCUG∼100 RNA toxicity perturbs several pathways including innate immunity, consistent with the observation in HD that immune activation precedes neuronal toxicity. Our results show that Drosophila rCAG.rCUG∼100 RNA toxicity is dependent upon Toll signaling and sensitive to autophagy, further implicating innate immune activation. In exhibiting molecular and cellular hallmarks of HD, double-stranded RNA-mediated activation of innate immunity is, therefore, a candidate pathway for this group of human genetic diseases.

High resolution spatial mapping of brominated pyrrole-2-aminoimidazole alkaloids distributions in the marine sponge Stylissa flabellata via MALDI-mass spectrometry imaging.

A number of pharmacologically active brominated pyrrole-2-aminoimidazole (B-P-2-AI) alkaloids have been isolated from several families of marine sponges, including those belonging to the genus Stylissa. In the present study, MALDI mass spectrometry imaging (MALDI-imaging) was applied to determine the spatial distribution of B-P-2-AIs within 20 µm cross sections of S. flabellata. A number of previously characterised B-P-2-AIs were readily identified by MALDI-imaging and confirmed by MS-MS and NMR profiling. Unknown B-P-2-AIs were also observed. Discrete microchemical environments were revealed for several B-P-2-AIs including dibromophakellin which was localised within the external pinacoderm and internal network of choanoderm chambers. Additionally, dibromopalau'amine and konbu'acidin B were also found to be confined to the choanoderm, while sceptrin was found to be highly abundant within the mesohyl. Further brominated compounds of unknown structure were also observed to have distinct localisation in both choanoderm chambers and the pinacoderm. These findings provide insights into the chemical ecology of S. flabellata, as most B-P-2-AIs were found on highly exposed surfaces, where they may act to prevent pathogens, predation and/or biofouling. Moreover this study demonstrates the power of MALDI-imaging to visualise the location of a range of metabolites in situ and to characterise compounds by MS-MS directly from intact specimens without the need for extraction. These methodologies facilitate selective targeting of micro-regions of sponge to screen for symbiotic microbial candidates or genes that may be involved in the production of the correlated compounds, and may represent a change in paradigm for natural product drug development.

Use of matrix-assisted laser desorption ionization-time of flight mass spectrometry to identify vancomycin-resistant enterococci and investigate the epidemiology of an outbreak.

The control of vancomycin-resistant enterococci (VRE) has become an increasing burden on health care resources since their discovery over 20 years ago. Current techniques employed for their detection include time-consuming and laborious phenotypic methods or molecular methods requiring costly equipment and consumables and highly trained staff. An accurate, rapid diagnostic test has the ability to greatly reduce the spread of this organism, which has the ability to colonize patients for long periods, potentially even lifelong. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a technology with the ability to identify organisms in seconds and has shown promise in the identification of other forms of antimicrobial resistance in other organisms. Here we show that MALDI-TOF MS is capable of rapidly and accurately identifying vanB-positive Enterococcus faecium VRE from susceptible isolates. Internal validation of the optimal model generated produced a sensitivity of 92.4% and a specificity of 85.2%. Prospective validation results, following incorporation into the routine laboratory work flow, demonstrated a greater sensitivity and specificity at 96.7% and 98.1%, respectively. In addition, the utilization of MALDI-TOF MS to determine the relatedness of isolates contributing to an outbreak is also demonstrated.

Development and validation of a novel molecular biomarker diagnostic test for the early detection of sepsis.

INTRODUCTION: Sepsis is a complex immunological response to infection characterized by early hyper-inflammation followed by severe and protracted immunosuppression, suggesting that a multi-marker approach has the greatest clinical utility for early detection, within a clinical environment focused on Systemic Inflammatory Response Syndrome (SIRS) differentiation. Pre-clinical research using an equine sepsis model identified a panel of gene expression biomarkers that define the early aberrant immune activation. Thus, the primary objective was to apply these gene expression biomarkers to distinguish patients with sepsis from those who had undergone major open surgery and had clinical outcomes consistent with systemic inflammation due to physical trauma and wound healing. METHODS: This was a multi-centre, prospective clinical trial conducted across four tertiary critical care settings in Australia. Sepsis patients were recruited if they met the 1992 Consensus Statement criteria and had clinical evidence of systemic infection based on microbiology diagnoses (n = 27). Participants in the post-surgical (PS) group were recruited pre-operatively and blood samples collected within 24 hours following surgery (n = 38). Healthy controls (HC) included hospital staff with no known concurrent illnesses (n = 20). Each participant had minimally 5 ml of PAXgene blood collected for leucocyte RNA isolation and gene expression analyses. Affymetrix array and multiplex tandem (MT)-PCR studies were conducted to evaluate transcriptional profiles in circulating white blood cells applying a set of 42 molecular markers that had been identified a priori. A LogitBoost algorithm was used to create a machine learning diagnostic rule to predict sepsis outcomes. RESULTS: Based on preliminary microarray analyses comparing HC and sepsis groups, a panel of 42-gene expression markers were identified that represented key innate and adaptive immune function, cell cycling, WBC differentiation, extracellular remodelling and immune modulation pathways. Comparisons against GEO data confirmed the definitive separation of the sepsis cohort. Quantitative PCR results suggest the capacity for this test to differentiate severe systemic inflammation from HC is 92%. The area under the curve (AUC) receiver operator characteristics (ROC) curve findings demonstrated sepsis prediction within a mixed inflammatory population, was between 86 and 92%. CONCLUSIONS: This novel molecular biomarker test has a clinically relevant sensitivity and specificity profile, and has the capacity for early detection of sepsis via the monitoring of critical care patients.

Perturbation of the Akt/Gsk3-ß signalling pathway is common to Drosophila expressing expanded untranslated CAG, CUG and AUUCU repeat RNAs.

Recent evidence supports a role for RNA as a common pathogenic agent in both the 'polyglutamine' and 'untranslated' dominant expanded repeat disorders. One feature of all repeat sequences currently associated with disease is their predicted ability to form a hairpin secondary structure at the RNA level. In order to investigate mechanisms by which hairpin-forming repeat RNAs could induce neurodegeneration, we have looked for alterations in gene transcript levels as hallmarks of the cellular response to toxic hairpin repeat RNAs. Three disease-associated repeat sequences--CAG, CUG and AUUCU--were specifically expressed in the neurons of Drosophila and resultant common transcriptional changes assessed by microarray analyses. Transcripts that encode several components of the Akt/Gsk3-ß signalling pathway were altered as a consequence of expression of these repeat RNAs, indicating that this pathway is a component of the neuronal response to these pathogenic RNAs and may represent an important common therapeutic target in this class of diseases.

Drosophila orthologue of WWOX, the chromosomal fragile site FRA16D tumour suppressor gene, functions in aerobic metabolism and regulates reactive oxygen species.

Common chromosomal fragile sites FRA3B and FRA16D are frequent sites of DNA instability in cancer, but their contribution to cancer cell biology is not yet understood. Genes that span these sites (FHIT and WWOX, respectively) are often perturbed (either increased or decreased) in cancer cells and both are able to suppress tumour growth. While WWOX has some tumour suppressor characteristics, its normal role and functional contribution to cancer has not been fully determined. We find that a significant proportion of Drosophila Wwox interactors identified by proteomics and microarray analyses have roles in aerobic metabolism. Functional relationships between Wwox and either CG6439/isocitrate dehydrogenase (Idh) or Cu-Zn superoxide dismutase (Sod) were confirmed by genetic interactions. In addition, altered levels of Wwox resulted in altered levels of endogenous reactive oxygen species. Wwox (like FHIT) contributes to pathways involving aerobic metabolism and oxidative stress, providing an explanation for the 'non-classical tumour suppressor' behaviour of WWOX. Fragile sites, and the genes that span them, are therefore part of a protective response mechanism to oxidative stress and likely contributors to the differences seen in aerobic glycolysis (Warburg effect) in cancer cells.