Administration of medicines to children: a practical guide.

Getting children to take medicines can be difficult. There is no 'one-size-fits-all' approach. When selecting medicines for children, it is important to consider the child's age, swallowing ability, ease of administration and accessibility of the product. Ask the child, parent or caregiver about their preference for formulations and flavours. There are different ways to alter the taste, aftertaste and mouth feel of medicines, which may help improve palatability. Pharmacists or medicines information services can assist with advice on suitable formulations or methods of administration.

Most Sinorhizobium meliloti Extracytoplasmic Function Sigma Factors Control Accessory Functions.

Bacteria must sense alterations in their environment and respond with changes in function and/or structure in order to cope. Extracytoplasmic function sigma factors (ECF σs) modulate transcription in response to cellular and environmental signals. The symbiotic nitrogen-fixing alphaproteobacterium Sinorhizobium meliloti carries genes for 11 ECF-like σs (RpoE1 to -E10 and FecI). We hypothesized that some of these play a role in mediating the interaction between the bacterium and its plant symbiotic partner. The bacterium senses changes in its immediate environment as it establishes contact with the plant root, initiates invasion of the plant as the root nodule is formed, traverses several root cell layers, and enters plant cortical cells via endocytosis. We used genetics, transcriptomics, and functionality to characterize the entire S. meliloti cohort of ECF σs. We discovered new targets for individual σs, confirmed others by overexpressing individual ECF σs, and identified or confirmed putative promoter motifs for nine of them. We constructed precise deletions of each ECF σ gene and its demonstrated or putative anti-σ gene and also a strain in which all 11 ECF σ and anti-σ genes were deleted. This all-ECF σ deletion strain showed no major defects in free-living growth, in Biolog Phenotype MicroArray assays, or in response to multiple stresses. None of the ECF σs were required for symbiosis on the host plants Medicago sativa and Medicago truncatula: the strain deleted for all ECF σ and anti-σ genes was symbiotically normal.IMPORTANCE Fixed (reduced) soil nitrogen plays a critical role in soil fertility and successful food growth. Much soil fertility relies on symbiotic nitrogen fixation: the bacterial partner infects the host plant roots and reduces atmospheric dinitrogen in exchange for host metabolic fuel, a process that involves complex interactions between the partners mediated by changes in gene expression in each partner. Here we test the roles of a family of 11 extracytoplasmic function (ECF) gene regulatory proteins (sigma factors [σs]) that interact with RNA polymerase to determine if they play a significant role in establishing a nitrogen-fixing symbiosis or in responding to various stresses, including cell envelope stress. We discovered that symbiotic nitrogen fixation occurs even when all 11 of these regulatory genes are deleted, that most ECF sigma factors control accessory functions, and that none of the ECF sigma factors are required to survive envelope stress.

Corrigendum: Characterization of Novel Plant Symbiosis Mutants Using a New Multiple Gene-Expression Reporter Sinorhizobium meliloti Strain.

[This corrects the article on p. 76 in vol. 9, PMID: 29467773.].

Characterization of Novel Plant Symbiosis Mutants Using a New Multiple Gene-Expression Reporter Sinorhizobium meliloti Strain.

The formation of nitrogen fixing root nodules by Medicago truncatula and Sinorhizobium meliloti requires communication between both organisms and coordinated differentiation of plant and bacterial cells. After an initial signal exchange, the bacteria invade the tissue of the growing nodule via plant-derived tubular structures, called infection threads. The bacteria are released from the infection threads into invasion-competent plant cells, where they differentiate into nitrogen-fixing bacteroids. Both organisms undergo dramatic transcriptional, metabolic and morphological changes during nodule development. To identify plant processes that are essential for the formation of nitrogen fixing nodules after nodule development has been initiated, large scale mutageneses have been conducted to discover underlying plant symbiosis genes. Such screens yield numerous uncharacterized plant lines with nitrogen fixation deficient nodules. In this study, we report construction of a S. meliloti strain carrying four distinct reporter constructs to reveal stages of root nodule development. The strain contains a constitutively expressed lacZ reporter construct; a PexoY-mTFP fusion that is expressed in infection threads but not in differentiated bacteroids; a PbacA-mcherry construct that is expressed in infection threads and during bacteroid differentiation; and a PnifH-uidA construct that is expressed during nitrogen fixation. We used this strain together with fluorescence microscopy to study nodule development over time in wild type nodules and to characterize eight plant mutants from a fast neutron bombardment screen. Based on the signal intensity and the localization patterns of the reporter genes, we grouped mutants with similar phenotypes and placed them in a developmental context.

The ROOT DETERMINED NODULATION1 gene regulates nodule number in roots of Medicago truncatula and defines a highly conserved, uncharacterized plant gene family.

The formation of nitrogen-fixing nodules in legumes is tightly controlled by a long-distance signaling system in which nodulating roots signal to shoot tissues to suppress further nodulation. A screen for supernodulating Medicago truncatula mutants defective in this regulatory behavior yielded loss-of-function alleles of a gene designated ROOT DETERMINED NODULATION1 (RDN1). Grafting experiments demonstrated that RDN1 regulatory function occurs in the roots, not the shoots, and is essential for normal nodule number regulation. The RDN1 gene, Medtr5g089520, was identified by genetic mapping, transcript profiling, and phenotypic rescue by expression of the wild-type gene in rdn1 mutants. A mutation in a putative RDN1 ortholog was also identified in the supernodulating nod3 mutant of pea (Pisum sativum). RDN1 is predicted to encode a 357-amino acid protein of unknown function. The RDN1 promoter drives expression in the vascular cylinder, suggesting RDN1 may be involved in initiating, responding to, or transporting vascular signals. RDN1 is a member of a small, uncharacterized, highly conserved gene family unique to green plants, including algae, that we have named the RDN family.

Transcript profiling in M. truncatula lss and sunn-1 mutants reveals different expression profiles despite disrupted SUNN gene function in both mutants.

A novel autoregulation of nodulation locus in Medicago truncatula, lss, silences the SUNN gene thorough a cis-acting mechanism. Microarray analysis was performed on the Affymetrix Gene Chip® Medicago Genome Array with cDNA isolated from seven-day-old seedlings of wild type, sunn-1 and lss plants. The results suggest that in lss plants expression of only a few dozen genes differs significantly from wild type while in sunn-1 plants expression of several hundred genes represented by over 800 probe sets is altered. These results suggest that the kinase domain modification caused by the sunn-1 mutation alters the receptor's influence on gene expression and that these differences are present even in the absence of nodulation.

The lss supernodulation mutant of Medicago truncatula reduces expression of the SUNN gene.

The number of nodules that form in a legume when interacting with compatible rhizobia is regulated by the plant. We report the identification of a mutant in nodule regulation in Medicago truncatula, like sunn supernodulator (lss), which displays shoot-controlled supernodulation and short roots, similar to sunn mutants. In contrast with the sunn-1 mutant, nodulation in the lss mutant is more extensive and is less sensitive to nitrate and ethylene, resembling the sunn-4 presumed null allele phenotype. Although the lss locus maps to the SUNN region of linkage group 4 and sunn and lss do not complement each other, there is no mutation in the genomic copy of the SUNN gene or in the 15-kb surrounding region in the lss mutant. However, expression of the SUNN gene in the shoots of lss plants is greatly reduced compared with wild-type plants. Analysis of cDNA from plants heterozygous for lss indicates that lss is a cis-acting factor affecting the expression of SUNN, and documented reversion events show it to be unstable, suggesting a possible reversible DNA rearrangement or an epigenetic change in the lss mutant. Assessment of the SUNN promoter revealed low levels of cytosine methylation in the 700-bp region proximal to the predicted transcription start site in both wild-type and lss plants, indicating that promoter hypermethylation is not responsible for the suppression of SUNN expression in lss. Thus, lss represents either a distal novel locus within the mapped region affecting SUNN expression or an uncharacterized epigenetic modification at the SUNN locus.

Conservation in function of a SCAR/WAVE component during infection thread and root hair growth in Medicago truncatula.

Nitrogen-fixing symbioses of plants are often associated with bacterially infected nodules where nitrogen fixation occurs. The plant host facilitates bacterial infection with the formation of infection threads, unique structures associated with these symbioses, which are invaginations of the host cell with the capability of traversing cellular junctions. Here, we show that the infection thread shares mechanistic similarities to polar-growing cells, because the required for infection thread (RIT) locus of Medicago truncatula has roles in root-hair, trichome, and infection-thread growth. We show that RIT encodes the M. truncatula ortholog of NAP1, a component of the SCAR/WAVE (suppressor of cAMP receptor/WASP-family verprolin homologous protein) complex that regulates actin polymerization, through the activation of ARP2/3. NAP1 of Arabidopsis thaliana functions equivalently to the M. truncatula gene, indicating that the mode of action of NAP1 is functionally conserved across species and that legumes have not evolved a unique functionality for NAP1 during rhizobial colonization. This work highlights the surprising commonality between polar-growing cells and a polar-growing cellular intrusion and reveals important insights into the formation and maintenance of infection-thread development.

cIAP1 cooperatively inhibits procaspase-3 activation by the caspase-9 apoptosome.

Although early studies of inhibitor of apoptosis proteins (IAPs) suggested that cIAP1 directly binds and inhibits caspases similarly to X-linked IAP (XIAP), a recent one found that micromolar concentrations of cIAP1 only weakly inhibit caspase-3, -7, or -9. Here, we show that cIAP1 specifically and cooperatively blocks the cytochrome c-dependent apoptosome in vitro. Hence, cIAP1 prevented the activation of procaspase-3 but had no effect on the processing of procaspase-9 or the activity of prior activated caspase-3. Like cIAP1, XIAP had no effect on procaspase-9 processing and was a more potent inhibitor of procaspase-3 activation than of already activated caspase-3 activity. Inhibition of procaspase-3 activation depended on BIR2 and BIR3 of cIAP1 and was independent of BIR1, RING, CARD, and UBA domains. Smac prevented cIAP1 from inhibiting procaspase-3 activation and reversed the inhibition by prior addition of cIAP1. A procaspase-9 mutant (D315A) that cannot produce the p12 subunit was resistant to inhibition by cIAP1. Therefore, the N-terminal Ala-Thr-Pro-Phe motif of the p12 subunit of the caspase-9 apoptosome facilitates apoptosome blockade. Consequently, cIAP1 cooperatively interacts with oligomerized processed caspase-9 in the apoptosome and blocks procaspase-3 activation.

Systematic review of case management.

The purpose of the present paper was to review the current state of evidence for types of case management, focusing on the last 10 years since publication of the Cochrane Systematic Reviews of case management and assertive community treatment. A literature review of electronic databases from 1995 to the present to identify recent research on psychiatric case management, both original studies and reviews, was carried out. Original articles were organized on basis of year of study, experimental group and outcome variables to determine patterns. Sixty relevant papers were located. Thirty-nine are reports of experimental trials of types of case management and 21 are reviews or discussion papers. The focus of research is on assertive community treatment or intensive case management, with only five papers on other forms of less intense case management. Numerous outcomes have been examined, of those examined often enough to draw meaningful conclusions only one, engagement with services, has been consistently positive. All other outcomes have produced mixed results. The strength of findings in favour of case management has weakened over time. A heterogeneous group of experimental designs limits comparisons. Numerous issues with methodology and definitions of types of case management have beset research in this field. Assertive types of case management (including assertive community treatment and intensive case management) are more effective than standard case management in reducing total number of days spent in hospital, improving engagement, compliance, independent living and patient satisfaction. More important than the type of service configuration is to understand the clinical criteria of the services provided and their effectiveness.

Nitrogen fixation mutants of Medicago truncatula fail to support plant and bacterial symbiotic gene expression.

The Rhizobium-legume symbiosis culminates in the exchange of nutrients in the root nodule. Bacteria within the nodule reduce molecular nitrogen for plant use and plants provide bacteria with carbon-containing compounds. Following the initial signaling events that lead to plant infection, little is known about the plant requirements for establishment and maintenance of the symbiosis. We screened 44,000 M2 plants from fast neutron-irradiated Medicago truncatula seeds and isolated eight independent mutant lines that are defective in nitrogen fixation. The eight mutants are monogenic and represent seven complementation groups. To monitor bacterial status in mutant nodules, we assayed Sinorhizobium meliloti symbiosis gene promoters (nodF, exoY, bacA, and nifH) in the defective in nitrogen fixation mutants. Additionally, we used an Affymetrix oligonucleotide microarray to monitor gene expression changes in wild-type and three mutant plants during the nodulation process. These analyses suggest the mutants can be separated into three classes: one class that supports little to no nitrogen fixation and minimal bacterial expression of nifH; another class that supports no nitrogen fixation and minimal bacterial expression of nodF, bacA, and nifH; and a final class that supports low levels of both nitrogen fixation and bacterial nifH expression.

Preservation of caspase-3 subunits from degradation contributes to apoptosis evoked by lactacystin: any single lysine or lysine pair of the small subunit is sufficient for ubiquitination.

Procaspase-3 (p32) is processed by upstream caspases to p12 and p20 subunits, which heterodimerize. Concomitant with formation of the active heterotetramer, p20 is autoprocessed to p17. Treatment of HL-60 cells with lactacystin, a selective inhibitor of the proteasome, exponentially increased caspase-3-like hydrolytic activity and induced apoptosis but had little or no effect on the activity of upstream caspase-8, caspase-9, or granzyme B. Lactacystin treatment decreased the p32 zymogen and evoked the accumulation of the p17 and p12 subunits. Treatment of transfected human retinoblast 911 cells with a proteasome inhibitor evoked the accumulation of epitope-tagged p12, p17, and p20 but had no effect on p32 zymogen. This result suggests that caspase-3 subunits, in contrast to the zymogen, are unstable because of degradation by the ubiquitin-proteasome system. Ubiquitin conjugates of p12 and p17 accumulated in cells that were cotransfected with p12 and a caspase inactive mutant of p17. Substitution of arginine for all eight lysines of p12 almost abolished its ubiquitination. Any single lysine or lysine pair was sufficient for p12 ubiquitination. Lactacystin treatment of HL-60 cells induced proteolytic processing of the X-linked inhibitor of apoptosis (XIAP) and decreased full-length XIAP, which is known to have ubiquitin-protein ligase activity for active caspase-3. These findings indicate that caspase-3 subunits can be degraded by the ubiquitin-proteasome system and suggest that lactacystin induces apoptosis in part by disabling the ubiquitin-protein ligase function of XIAP and by stabilizing active caspase-3 subunits.

Caspase processing activates atypical protein kinase C zeta by relieving autoinhibition and destabilizes the protein.

Treatment of HeLa cells with tumour necrosis factor alpha (TNFalpha) induced caspase processing of ectopic PKC (protein kinase C) zeta, which converted most of the holoenzyme into the freed kinase domain and increased immune-complex kinase activity. The goal of the present study was to determine the basis for the increased kinase activity that is associated with caspase processing of PKC zeta. Atypical PKC iota is largely identical with PKC zeta, except for a 60-amino-acid segment that lacks the caspase-processing sites of the zeta isoform. Replacement of this segment of PKC zeta with the corresponding segment of PKC iota prevented caspase processing and activation of the kinase function. Processing of purified recombinant PKC zeta by caspase 3 in vitro markedly increased its kinase activity. Caspase processing activated PKC zeta in vitro or intracellularly without increasing the phosphorylation of Thr410 of PKC zeta, which is required for catalytic competency. The freed kinase domain of PKC zeta had a much shorter half-life than the holoenzyme in transfected HeLa cells and in non-transfected kidney epithelial cells. Treatment with TNF-alpha shortened the half-life of the kinase domain protein, and proteasome blockade stabilized the protein. Studies of kinase-domain mutants indicate that a lack of negative charge at Thr410 can shorten the half-life of the freed kinase domain. The present findings indicate that the freed kinase domain has substantially higher kinase activity and a much shorter half-life than the holoenzyme because of accelerated degradation by the ubiquitin-proteasome system.

Dual genetic pathways controlling nodule number in Medicago truncatula.

We report the isolation and characterization of a new Medicago truncatula hyper-nodulation mutant, designated sunn (super numeric nodules). Similar to the previously described ethylene-insensitive mutant sickle, sunn exhibits a 10-fold increase in the number of nodules within the primary nodulation zone. Despite this general similarity, these two mutants are readily distinguished based on anatomical, genetic, physiological, and molecular criteria. In contrast to sickle, where insensitivity to ethylene is thought to be causal to the hyper-nodulation phenotype (R.V. Penmetsa, D.R. Cook [1997] Science 275: 527-530), nodulation in sunn is normally sensitive to ethylene. Nevertheless, sunn exhibits seedling root growth that is insensitive to ethylene, although other aspects of the ethylene triple response are normal; these observations suggest that hormonal responses might condition the sunn phenotype in a manner distinct from sickle. The two mutants also differ in the anatomy of the nodulation zone: Successful infection and nodule development in sunn occur predominantly opposite xylem poles, similar to wild type. In sickle, however, both infection and nodulation occur randomly throughout the circumference of the developing root. Genetic analysis indicates that sunn and sickle correspond to separate and unlinked loci, whereas the sunn/skl double mutant exhibits a novel and additive super-nodulation phenotype. Taken together, these results suggest a working hypothesis wherein sunn and sickle define distinct genetic pathways, with skl regulating the number and distribution of successful infection events, and sunn regulating nodule organogenesis.

Lack of constitutive activity of the free kinase domain of protein kinase C zeta. Dependence on transphosphorylation of the activation loop.

Following the induction of apoptosis in mammalian cells, protein kinase C zeta (PKC zeta) is processed between the regulatory and catalytic domains by caspases, which increases its kinase activity. The catalytic domain fragments of PKC isoforms are considered to be constitutively active, because they lack the autoinhibitory amino-terminal regulatory domain, which includes a pseudosubstrate segment that plugs the active site. Phosphorylation of the activation loop at Thr(410) is known to be sufficient to activate the kinase function of full-length PKC zeta, apparently by inducing a conformational change, which displaces the amino-terminal pseudosubstrate segment from the active site. Amino acid substitutions for Thr(410) of the catalytic domain of PKC zeta (CAT zeta) essentially abolished the kinase function of ectopically expressed CAT zeta in mammalian cells. Similarly, substitution of Ala for a Phe of the docking motif for phosphoinositide-dependent kinase-1 prevented activation loop phosphorylation and abolished the kinase activity of CAT zeta. Treatment of purified CAT zeta with the catalytic subunit of protein phosphatase 1 decreased activation loop phosphorylation and kinase activity. Recombinant CAT zeta from bacteria lacked detectable kinase activity. Phosphoinositide-dependent kinase-1 phosphorylated the activation loop and activated recombinant CAT zeta from bacteria. Treatment of HeLa cells with fetal bovine serum markedly increased the phosphothreonine 410 content of CAT zeta and stimulated its kinase activity. These findings indicate that the catalytic domain of PKC zeta is intrinsically inactive and dependent on the transphosphorylation of the activation loop.