Evaluation of early clinical failure criteria for gram-negative bloodstream infections.

OBJECTIVES: The aim was the development of early clinical failure criteria (ECFC) to predict unfavourable outcomes in patients with Gram-negative bloodstream infections (GN-BSI). METHODS: Adults with community-onset GN-BSI who survived hospitalization for ≥72 hr at Prisma Health-Midlands hospitals in Columbia, SC, USA from January 1, 2010 to June 30, 2015 were identified. Multivariable logistic regression was used to examine the association between clinical variables between 72 and 96 hr after GN-BSI and unfavourable outcomes (28-day mortality or hospital length of stay >14 days from GN-BSI onset). RESULTS: Among 766 patients, 225 (29%) had unfavourable outcomes. After adjustments for Charlson Comorbidity Index and appropriateness of empirical antimicrobial therapy in multivariable model, predictors of unfavourable outcomes included systolic blood pressure <100 mmHg or vasopressor use (adjusted odds ratio (aOR) 1.8, 95% confidence interval (CI) 1.2-2.9), heart rate >100 beats/minute (aOR 1.7, 95% CI 1.1-2.5), respiratory rate ≥22 breaths/minute or mechanical ventilation (aOR 2.1, 95% CI 1.4-3.3), altered mental status (aOR 4.5, 95% CI 2.8-7.1), and white blood cell count >12 000/mm3 (aOR 2.7, 95% CI 1.8-4.1) between 72 and 96 hr after index GN-BSI. Area under receiver operating characteristic curve of ECFC model in predicting unfavourable outcomes was 0.77 (0.84 and 0.71 in predicting 28-day mortality and prolonged hospitalization, respectively). CONCLUSIONS: Risk of 28-day mortality or prolonged hospitalization can be estimated between 72 and 96 hr after GN-BSI using ECFC. These criteria may have clinical utility in management of GN-BSI and may improve methodology of future investigations assessing response to antimicrobial therapy based on a standard evidence-based definition of early clinical failure.

Enhanced beta cell proliferation in mice overexpressing a constitutively active form of Akt and one allele of p21Cip.

AIMS/HYPOTHESIS: The ability of pancreatic beta cells to proliferate is critical both for normal tissue maintenance and in conditions where there is an increased demand for insulin. Protein kinase B(Akt) plays a major role in promoting proliferation in many cell types, including the insulin-producing beta cells. We have previously reported that mice overexpressing a constitutively active form of Akt(caAkt (Tg)) show enhanced beta cell proliferation that is associated with increased protein levels of cyclin D1, cyclin D2 and cyclin-dependent kinase inhibitor 1A (p21(Cip)). In the present study, we sought to assess the mechanisms responsible for augmented p21(Cip) levels in caAkt(Tg) mice and test the role of p21(Cip) in the proliferative responses induced by activation of Akt signalling. METHODS: To gain a greater understanding of the relationship between Akt and p21(Cip), we evaluated the mechanisms involved in the modulation of p2(Cip) by Akt and the in vivo role of reduced p21(Cip) in proliferative responses induced by Akt. RESULTS: Our experiments showed that Akt signalling regulates p21(Cip) transcription and protein stability. caAkt(Tg) /p21(Cip+/-) mice exhibited fasting and fed hypoglycaemia as well as hyperinsulinaemia when compared with caAkt(Tg) mice. Glucose tolerance tests revealed improved glucose tolerance in caAkt(Tg)/p21(Cip+/-) mice compared with caAkt (Tg). These changes resulted from increased proliferation, survival and beta cell mass in caAkt(Tg)/p21(Cip+/-) compared with caAkt(Tg) mice. CONCLUSIONS/INTERPRETATION: Our data indicate that increased p21(Cip) levels in caAkt(Tg) mice act as a compensatory brake, protecting beta cells from unrestrained proliferation. These studies imply that p21(Cip) could play important roles in the adaptive responses of beta cells to proliferate in conditions such as in insulin resistance.

Applying an adaptive watershed to the tissue cell quantification during T-cell migration and embryonic development.

Cell and particle quantification is one of the frequently used techniques in biology and clinical study. Variations of cell/particle population and/or protein expression level can provide information on many biological processes. In this chapter, we propose an image-based automatic quantification approach that can be applied to images from both fluorescence and electron microscopy. The algorithm uses local maxima to identify labelling targets and uses watershed segmentation to define their boundaries. The method is able to provide information on size, intensity centroids and average intensity within the labelling partitions. Further developed from this method, we demonstrated its applications in four different research projects, including recruitment enumeration of circulating T cell in non-lymphoid tissues, cell clustering in the early development of the chick embryo, gold particle localization and clustering in electron microscopy, and registration/co-localization of transcription factors in neural tube development of early chick embryo. The advantages and limitations of the method are also discussed.

A novel family of single VWC-domain proteins in invertebrates.

Using a combination of sequence analysis tools, a novel family of 13 short Drosophila melanogaster proteins with similarity to a single von Willebrand factor C-domain (SVC proteins) has been identified. SVCs are predicted to be secreted and a structural model for the family is proposed, using a known von Willebrand factor C-domain (VWC) structure as template. All SVCs possess eight cysteines, consistent with the loss of one bonded pair from the 10-cysteine canonical pattern of most VWC domains. Unlike most Drosophila genes, many SVCs are not expressed during development, and misexpression has no apparent effect on development or viability. SVCs appear to be restricted to arthropods. A role for SVCs in response to environmental challenges is proposed.

Insect oenocytes: a model system for studying cell-fate specification by Hox genes.

During insect development, morphological differences between segments are controlled by the Hox gene family of transcription factors. Recent evidence also suggests that variation in the regulatory elements of these genes and their downstream targets underlies the evolution of several segment-specific morphological traits. This review introduces a new model system, the larval oenocyte, for studying the evolution of fate specification by Hox genes at single-cell resolution. Oenocytes are found in a wide range of insects, including species using both the short and the long germ modes of development. Recent progress in our understanding of the genetics and cell biology of oenocyte development in the fruitfly Drosophila melanogaster is discussed. In the D. melanogaster embryo, the formation of this cell type is restricted to the first 7 abdominal segments and is under Hox gene control. Oenocytes delaminate from the dorsal ectoderm of A1-A7 in response to an induction that involves the epidermal growth factor receptor (EGFR) signalling pathway. Although the receptor itself is required in the presumptive oenocytes, its ligand Spitz (Spi) is secreted by a neighbouring chordotonal organ precursor (COP). Thus, in dorsal regions, local signalling from this component of the developing peripheral nervous system induces the formation of oenocytes. In contrast, in lateral regions of the ectoderm, Spi signal from a different COP induces the formation of secondary COPs in a homeogenetic manner. This dorsoventral difference in the fate induced by Spi ligand is controlled by a prepattern in the responding ectoderm that requires the Spalt (Sal) transcription factor. Sal protein is expressed in the dorsal but not lateral ectoderm and acts as a competence modifier to bias the response to Spi ligand in favour of the oenocyte fate. We discuss a recently proposed model that integrates the roles of Sal and the EGFR pathway in oenocyte/chordotonal organ induction. This model should provide a useful starting point for future comparative studies of these ectodermal derivatives in other insects.

spalt-dependent switching between two cell fates that are induced by the Drosophila EGF receptor.

Signaling from the EGF receptor (EGFR) can trigger the differentiation of a wide variety of cell types in many animal species. We have explored the mechanisms that generate this diversity using the Drosophila peripheral nervous system. In this context, Spitz (SPI) ligand can induce two alternative cell fates from the dorsolateral ectoderm: chordotonal sensory organs and non-neural oenocytes. We show that the overall number of both cell types that are induced is controlled by the degree of EGFR signaling. In addition, the spalt (sal) gene is identified as a critical component of the oenocyte/chordotonal fate switch. Genetic and expression analyses indicate that the SAL zinc-finger protein promotes oenocyte formation and supresses chordotonal organ induction by acting both downstream and in parallel to the EGFR. To explain these findings, we propose a prime-and-respond model. Here, sal functions prior to signaling as a necessary but not sufficient component of the oenocyte prepattern that also serves to raise the apparent threshold for induction by SPI. Subsequently, sal-dependent SAL upregulation is triggered as part of the oenocyte-specific EGFR response. Thus, a combination of SAL in the responding nucleus and increased SPI ligand production sets the binary cell-fate switch in favour of oenocytes. Together, these studies help to explain how one generic signaling pathway can trigger the differentiation of two distinct cell types.

The differentiation of the serotonergic neurons in the Drosophila ventral nerve cord depends on the combined function of the zinc finger proteins Eagle and Huckebein.

The Drosophila ventral nerve cord (vNC) derives from a stereotyped population of neural stem cells, neuroblasts (NBs), each of which gives rise to a characteristic cell lineage. The mechanisms leading to the specification and differentiation of these lineages are largely unknown. Here we analyse mechanisms leading to cell differentiation within the NB 7-3 lineage. Analogous to the grasshopper, NB 7-3 is the progenitor of the Drosophila vNC serotonergic neurons. The zinc finger protein Eagle (Eg) is expressed in NB 7-3 just after delamination and is present in all NB 7-3 progeny until late stage 17. DiI cell lineage tracing and immunocytochemistry reveal that eg is required for normal pathfinding of interneuronal projections and for restricting the cell number in the thoracic NB 7-3 lineage. Moreover, eg is required for serotonin expression. Ectopic expression of Eg protein forces specific additional CNS cells to enter the serotonergic differentiation pathway. Like NB 7-3, the progenitor(s) of these ectopic cells express Huckebein (Hkb), another zinc finger protein. However, their progenitors do not express engrailed (en) as opposed to the NB 7-3 lineage, where en acts upstream of eg. We conclude that eg and hkb act in concert to determine serotonergic cell fate, while en is more distantly involved in this process by activating eg expression. Thus, we provide the first functional evidence for a combinatorial code of transcription factors acting early but downstream of segment polarity genes to specify a unique neuronal cell fate.

The cell adhesion molecule, connectin, and the development of the Drosophila neuromuscular system.

The connectin gene of Drosophila has been identified as a candidate direct target of homeotic gene control and has also been implicated in the formation of specific neuromuscular connections. The gene product, connectin, is a member of the leucine-rich repeat protein family and we show that it is attached to the cell surface via a glycosylphosphatidylinositol linkage and that it can mediate homotypic cell-cell adhesion in vitro. The expression of connectin protein during Drosophila embryogenesis provides support for a role in adhesion in vivo. In the central nervous system, it is initially expressed on longitudinal glia and on a few identified neurons. These cells extend processes and connect up to form a continuous scaffold of connectin-expressing cells, presaging the development of axonal pathways. Later, connectin is expressed on specific axons as they track along the connectin scaffold. Glial expression then declines and connectin appears on axons that fasciculate with pre-existing connectin-positive bundles. Thus scaffold formation, axon pathfinding and fasciculation involve specific contacts between connectin-positive cells. The timing and pattern of connectin expression suggest that it may play an important role in mediating specific interactions through homotypic cell adhesion.

Connectin, a target of homeotic gene control in Drosophila.

The homeotic genes of Drosophila encode transcription factors that specify morphological differences between segments. To identify the genes that they control, we developed a chromatin immunopurification approach designed to isolate in vivo binding sites for the products of the homeotic gene Ultrabithorax. Here, we report the analysis of one immunopurified binding site. This 110 bp fragment maps within a regulatory region of a gene under homeotic control, connectin. A 4 kb DNA fragment, including the immunopurified binding site, is sufficient to reproduce the appropriate homeotic control within a subset of the full tissue distribution of connectin. Analysis of the role of the 110 bp binding site indicates that it mediates transcriptional controls by Ultrabithorax and other homeotic genes. This is the first report of a functional in vivo binding site isolated using the chromatin immunopurification method. We also show that the protein product of the connectin gene is predicted to be a cell-surface molecule containing leucine-rich repeats. The protein, connectin, can mediate cell-cell adhesion thus suggesting a direct link between homeotic gene function and processes of cell-cell recognition.

Targets of homeotic gene regulation in Drosophila.

We have used a chromatin immunopurification approach to identify target genes regulated by the homeotic gene Ultrabithorax. A monoclonal antibody against the Ultrabithorax gene product is used to immunopurify in vivo Ultrabithorax protein binding sites in embryonic chromatin. The procedure gives an enrichment of sequences with matches to a consensus homeodomain binding site. In one case we have shown that an immunopurified sequence lies within a 4 kb fragment that acts in vivo as a homeotic response element. We anticipate that this approach will enable us to identify further targets, allowing the analysis of their regulation and function. The chromatin immunopurification strategy may be of general application for the identification of direct in vivo targets of DNA-binding proteins.

Blocking cell division does not remove the requirement for Polycomb function in Drosophila embryogenesis.

The Polycomb (Pc) gene is required from the extended germ band stage onwards, to maintain spatially restricted patterns of homeotic gene expression. It has been thought to be involved in the 'stable inheritance of the determined state'. In this paper, we have tested the notion that the Pc gene is required specifically during or after DNA replication to enable the stable transmission of states of gene activity. We found that arresting cell division using the string mutation or blocking DNA replication with aphidicolin failed to prevent ectopic expression of the homeotic gene Ultrabithorax in Pc mutants. Thus, even in the absence of DNA replication, Pc is required to maintain spatially restricted patterns of homeotic gene expression. The role of the Pc gene product in the stable repression of homeotic gene transcription is discussed.

Targets of homeotic gene control in Drosophila.

The homeotic gene Ultrabithorax (Ubx) encodes homeodomain-containing transcription factors that determine segmental identity in Drosophila. Here, an immunopurification procedure is described that enriches for embryonic chromatin fragments containing binding sites for Ubx protein. In two cases these binding sites are located near embryonic transcription units regulated by the Ubx locus in vivo. Thus, these transcripts may correspond to Ubx target genes involved in elaborating segment-specific developmental pathways.

California encephalitis virus prevalence throughout the Yukon Territory, 1971-1974.

California encephalitis (CE) virus (snowshoe hare subtype) was isolated from 1 of 38 pools comprising 970 unengorged female Aedes canadensis mosquitoes and from 3 of 152 pools containing 5,676 A. communis mosquitoes which were collected in the Yukon Territory, Canada between latitudes 61 and 66 degrees N during June and July 1974. During four summers 1971 through 1974, this virus was recovered from 26 of 648 pools derived from 30,686 mosquitoes of 4 species. Isolation of CE virus from 1 of 109 pools of Aedes sp. larvae collected during May 1974 suggests maintenance of this virus over winter by transovarial transfer. Infectivity has been maintained in Culiseta inornata mosquitoes which were held continuously at 32 degrees F for 138 days. Neutralizing antibodies to CE virus were detected in 705 of 4,913 (14%) mammals collected during summers 1971 through 1974, including 430 of 1,076 (40%) snowshoe hares (Lepus americanus), 266 of 3,610 (7%) ground squirrels (Citellus undulatus) and 9 of 227 (4%) red squirrels (Tamiasciurus hudsonicus).