Concordance between epidemiological evaluation of probability of transmission and whole genome sequence relatedness among hospitalized patients acquiring Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae.

OBJECTIVES: We aimed to evaluate the concordance between epidemiologically determined transmission and genetic linkage of Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp). METHODS: We included consecutive KPC-Kp carriers between December 2016 and April 2017 in a hospital endemic for KPC-Kp. We assessed epidemiological relatedness between patients by prospective investigations by the infection control team. The probability of epidemiological relatedness was classified into four groups: no suspected transmission, low, moderate and high probability of transmission. Whole-genome sequencing of isolates was performed. Genetic linkage between KPC-Kp isolates was expressed by distance between isolates in single nucleotide polymorphisms (SNPs). We established an SNP cut-off defining a different strain based on the reconstructed phylogenetic tree. We compared the epidemiological and genetic linkage of all isolates from all patients. RESULTS: The study included 25 KPC-Kp carriers with 49 isolates. SNP variance was available for 1129 crossed patient-isolate pairs. Genomic linkage, based on a cut-off of 80 SNPs to define related isolates, was found in 115/708 (16.2%) of isolates with no transmission suspected epidemiologically, 27/319 (8.5%) of low, 11/26 (42.3%) of moderate and 64/76 (84.2%) of high epidemiological transmission risk determination (p < 0.001 for trend). Similar results and significant trends were shown on sensitivity analyses using a lower SNP cut-off (six SNPs) and patient-isolate unique pairs, analysing the first isolate from each patient. CONCLUSIONS: While significant concordance between epidemiological and genomic transmission patterns was found, epidemiological investigations of transmission are limited by the possibility of unidentified transmissions or over-estimation of associations. Genetic linkage analysis is an important aid to epidemiological transmission assessment.

Systematic review of spica casting for the treatment of paediatric diaphyseal femur fractures.

PURPOSE: Paediatric femur fractures are commonly encountered and often successfully managed with spica casting. Despite spica casting's long history there is little formal guidance for optimal outcomes and no consolidation of existing literature. The purpose of this study is to review the available literature regarding the use of spica casting for the management of paediatric diaphyseal femur fractures. METHODS: The PubMed database was queried for all research articles including the phrase "spica". A total of 788 abstracts were reviewed for relevance to the current study. Data was extracted from all available research studies which specified tolerance for fracture angulation or shortening in the cast. Additionally, all articles describing alternative materials, methods for spica application, and complications of spica casting were reviewed. RESULTS: In all, 106 articles were found relevant to the management of diaphyseal femur fractures in the paediatric population. The aggregated, accepted fracture shortening decreased from 16 mm to 18 mm before age ten years to 12 mm to 14 mm after puberty. Aggregated, accepted angulation decreased from 14° to 16° varus/valgus and 18° to 22° pro/recurvatum before age two years, to 6° to 8° and 10° to 12° by puberty, respectively. The overall reported complication rate was 19.6%, with the most common complication being skin compromise in 8.2% of patients, followed by unacceptable angulation at the fracture site in 4.2% of patients and excessive limb shortening in 1.9% of patients. CONCLUSION: This article reviews the available spica casting literature and compiles the available data. Spica casting offers a safe, effective means for definitive management of paediatric diaphyseal femur fractures. Future research identifying the rate and pattern of remodelling as it relates to angulation and shortening at various patient ages, particularly beyond the aforementioned norms, would be valuable to identify true biological tolerances versus accepted expert opinion. LEVEL OF EVIDENCE LEVEL II: Review of level II evidence.

Binding of straight-chain saturated dicarboxylic acids to albumin.

Dicarboxylic acids are prominent features of several diseases, including Reye's syndrome. Long-chain dicarboxylic acids have profound effects on the function and structure of isolated mitochondria, suggesting that they could contribute to the mitochondrial dysfunction in Reye's syndrome. Binding of fatty acids to albumin and the intracellular fatty acid-binding proteins is important in regulating the transport and metabolism of fatty acids and protects against the toxic effects of unbound fatty acids. We studied the binding of dicarboxylic acids to defatted albumin using equilibrium dialysis to assess to what extent dicarboxylic acids are likely to be bound in the plasma of patients. Dicarboxylic acids bind weakly to albumin in a molar ratio of 3.8, 4.2, 1.6, 0.8, and 0.7 to 1 for octadecanedioic, hexadecanedioic, tetradecanedioic, dodecanedioic, and decanedioic acid, respectively. The dissociation constants for long-chain dicarboxylic acids are 100-1,000-fold larger than those of comparable monocarboxylic acids. Oleate competes with dicarboxylic acid and reduces the moles of dicarboxylic acid bound per mol of albumin to less than 1. Octanoate inhibits dicarboxylic acid binding. Our observations indicate that in Reye's syndrome, substantial concentrations of dicarboxylic acids of patients may be free and potentially toxic to mitochondria and other cellular processes.

Multiple protein complexes, including AP2 and Sp1, interact with a specific site within the rat preprotachykinin-A promoter.

We demonstrate that there is a unique AP2 binding site in the rat preprotachykinin-A promoter (rPPT) spanning -865 to -47. AP2 is a transcription factor whose expression in sensory neurons has been correlated with rPPT expression in these cells. This binding site is adjacent to an element we previously identified as binding a single stranded DNA binding protein which was also present in sensory neurons. These two complexes encompass a region which we had proposed might form a stem-loop structure, allowing binding of the single stranded DNA binding protein to the DNA. Here using electrophoretic mobility shift analysis we demonstrate that the DNA region corresponding to the putative stem-loop structure is bound by a variety of transcription factors, including in addition to AP2 the ubiquitous Sp1. DNase 1 footprint analysis demonstrates that binding to this domain by the proteins recognising the double-stranded form of the cis acting element is mutually exclusive. A promoter fragment containing this domain demonstrated a DNase 1 footprint over the 5' region of the stem-loop structure. Competition of the binding for this element by an oligonucleotide corresponding to the stem-loop structure removed the 5' footprint and exposed a new footprint over the 3' region of the stem-loop structure and extending for several base pairs. This change in protection observed with DNase 1 digestion also correlated with changes of the DNase 1 pattern at specific locations 3' of the proposed stem-loop structure. These changes correlated with two DNA sequences which were homologous to one another and to a region within the proposed stem-loop structure. Our results indicate that AP2 could regulate rPPT gene expression by a variety of mechanisms.

A review and reevaluation of the role of serotonin in the modulation of lordosis behavior in the female rat.

The role of serotonin (5-HT) in the modulation of sexual receptivity (lordosis) in the female rat is reviewed and reevaluated. The effects on lordosis of drug treatments that decrease or increase the activity and availability of central 5-HT are first discussed, and this is followed by an evaluation of the effects of drugs that act directly at 5-HT receptors. In order to shed light on the physiological significance of effects of serotonergic drugs on lordosis, there is also a review of what is known of changes in levels of serotonergic activity and densities of 5-HT receptors in the female rat brain that take place through the estrous cycle and in response to administration of behaviorally effective doses of gonadal steroids. Serotonin has generally been thought to have a tonic, inhibitory effect on lordosis. However, it is concluded that 5-HT can either inhibit or facilitate lordosis depending on which subtypes of central 5-HT receptors become activated. Because of a lack of consistent or compelling evidence of effects of ovarian hormones on serotonergic activity or 5-HT receptors in critical areas of the brain, it is stated that there is at present no basis to conclude that the effects of pharmacological manipulations of serotonergic activity on lordosis reflect an important, physiological role of 5-HT in the modulation of lordosis behavior in the female rat.

Human immunodeficiency virus wasting syndrome may represent a treatable myopathy.

We evaluated 5 patients with the diagnosis of HIV wasting syndrome. None had severe diarrhea or other causes for malabsorption. All had myopathy by clinical, laboratory, and muscle biopsy criteria. Withdrawal of azidothymidine in 3 patients did not lead to improvement. Corticosteroid therapy was effective in 3 patients.

Sex differences in the effects of 1-(m-trifluoromethylphenyl) piperazine and 1-(m-chlorophenyl) piperazine on copulatory behavior in the rat.

Peripheral administrations of TFMPP (0.2- 1 mg/kg) or MCPP (1 mg/kg) facilitated lordosis behavior in female rats treated with estradiol benzoate, and had no effects in females primed with estradiol benzoate and progesterone. In contrast, TFMPP (1 mg/kg) and MCPP (1 mg/kg) inhibited copulatory behavior in male rats. It is concluded that there are sex differences in the effects of TFMPP and MCPP on copulatory behavior in the rat. Moreover, it is suggested that the effects of these drugs on copulatory behavior may be mediated by activation of 5-HT1B and/or 5-HT1C receptors, or by blockade of activity at 5-HT3 receptors.

Methysergide inhibits and facilitates lordosis behavior in the female rat in a time-dependent manner.

Reports in the literature have indicated a facilitatory effect of the serotonin antagonist methysergide on lordosis behavior, suggesting an inhibitory role for serotonergic activity. In the present series of experiments, methysergide (7 mg/kg) was found to inhibit lordosis behavior 30 min after intraperitoneal administration to females, treated chronically with estradiol benzoate, or acutely with estradiol benzoate and progesterone. However, methysergide was found to facilitate lordosis behavior 200 and 300 min after administration to female rats treated acutely with estradiol benzoate. These data suggest a time-dependent inhibitory effect of methysergide, and are consistent with the hypothesis that the activity of serotonin type 2 receptors facilitates lordosis behavior in the female rat.

Three immediate early gene response elements in the proximal preprotachykinin-A promoter in two functionally distinct domains.

The preprotachykinin-A promoter contains two blocks of DNA sequence, with a high degree of homology to one another, both containing activator protein 1/cAMP response element-like elements which constitute cis-acting regulatory domains. These two domains are differentially regulated in HeLa cells and primary cultures of dorsal root ganglion neurons when they are placed in the context of a reporter gene driven by the c-fos minimum promoter. One of the domains, corresponding to a region of the preprotachykinin promoter spanning nucleotides -345 to -308, contains two activator protein 1 elements adjacent to an E-box binding protein consensus sequence. Both of the activator protein 1 elements can bind a complex containing c-fos/c-fos related antigen proteins and the adjacent E-box element is specifically recognized by proteins present in HeLa nuclear extract. This domain requires the synergistic action of both activator protein 1 elements to drive expression of the reporter gene in both HeLa and dorsal root ganglion cells. The second or proximal domain spans nucleotides -198 to -155 and contains a previously characterized activator protein 1/cAMP response element/ATF enhancer element which, in contrast to the activator protein 1 elements in the distal domain, functions in both HeLa and dorsal root ganglion cells as one copy. This domain is differentially regulated in HeLa and dorsal root ganglia. The previously characterized enhancer activity is repressed in the context of the extended cis-acting domain in HeLa cells but remains active in dorsal root ganglion, although no further enhancement of activity supported by the single enhancer is observed when in the context of the extended sequence. This proximal domain, in addition to binding the enhancer complex, can be bound by at least two other complexes, one of which binds to an E-box consensus sequence. As the elements corresponding to the E-box consensus in both domains cross-compete for binding of specific complex(es) it would appear that repression of the activity of the proximal domain is correlated with a specific protein complex binding adjacent to the characterized enhancer in the region spanning nucleotides -198 to -155. The preprotachykinin-A proximal promoter is therefore bound by multiple activator protein I complexes, which in the context of the cis-acting domains in which they are present can be differentially regulated. In the proximal domain their function may also be regulated in a tissue-specific manner by other proteins which bind to adjacent regulatory elements.

Repression of preprotachykinin-A promoter activity is mediated by a proximal promoter element.

The rat preprotachykinin-A promoter, which is able to direct reporter gene expression in adult dorsal root ganglia neurons grown in culture, has no detectable activity in HeLa and PC12 cells. DNAase 1 footprinting and electrophoretic mobility shift analyses with HeLa nuclear extract indicated the presence of a protein complex binding to a region of the rat preprotachykinn-A gene promoter between the TATA box and the major transcriptional start site. We demonstrate that the sequence of the preprotachykinin-A promoter spanning nucleotides -47 to +92 functions to repress reporter gene expression in HeLa and PC12 cells but not in adult rat dorsal root ganglia grown in culture, and that this repression is correlated with a protein(s) binding to the element between the TATA box and major transcription initiation site. These results indicate that the tissue-specific expression of the preprotachykinin-A gene could require the interaction of both positive and negative regulatory DNA elements.

The nuclear autoimmune antigen Ku is also present on the cell surface.

Polyclonal antibodies were raised against the individual 85 and 70 kDa subunits of the Ku complex purified from nuclear extract prepared from the T cell line MLA144. They specifically recognise the appropriate subunits of the Ku complex from whole cell extract of HeLa cells using Western blot analysis. They are also able to identify the Ku proteins present in the cell membrane using FACS analysis.

A correlational and factor analysis of anticipatory and consummatory measures of sexual behavior in the male rat.

This study investigated the relationship among measures of anticipatory and consummatory sexual behavior displayed by male rats in the bilevel chambers designed by Mendelson and Gorzalka (1987). Normative data from a standard test of sexual behavior were gathered from 80 intact, sexually experienced male Long-Evans rats and subjected to multiple correlational and factor analyses. The correlational analysis confirmed that several consummatory measures of copulation were related significantly, whereas the anticipatory measure, level changing, was statistically independent of consummatory measures. Factor analysis using orthogonal rotations revealed five factors that accounted for 95% of the intersubject variance for all measures: Copulatory Rate, Initiation, Hit Rate, Mount Count, and Anticipation. These results indicate that at least five conceptual mechanisms are required in any theoretical description of male sexual behavior in the bilevel chamber. In particular, the extraction of separate anticipation and initiation factors indicates that these aspects of male sexual behavior are distinct. The use of bilevel chambers thus may facilitate the identification of potential neurochemical or endocrine mechanisms associated with different aspects of male sexual motivation. In addition, several statistical techniques are discussed with the aim of reducing the elevated experiment-wise error that can occur when related measures of sexual behavior are analyzed independently.

An NGF-inducible octamer binding protein activity in a C1300 neuroblastoma cell line.

Nerve growth factor (NGF) induces a protein in the C1300 mouse neuroblastoma cell line which recognises the octamer DNA consensus sequence 'ATGCAAAT'. This protein is absent, or only minimally present, in C1300 cells prior to induction with NGF. This induced octamer binding protein is detectable by gel retardation analysis within 3 h of NGF treatment and increases progressively with 24 h and 72 h of exposure to NGF.

A role for the octamer-binding protein in preprotachykinin-A gene expression.

A rat Preprotachykinin-A promoter fragment has been previously identified which supports reporter gene activity in primary cultures of adult dorsal root ganglion neurons. That study demonstrated that two promoter domains which exhibit enhancer activity in these neurons are bound by the same classes of transcription factors. Further, the two domains exhibit similarities with respect to the relationship of bound transcription factors within each domain. This suggests that these domains may function in an identical manner or may act synergistically to regulate gene expression. These domains contain recognition motifs for at least three classes of transcription factors: octamer-binding proteins, Sp1-related proteins and an as yet unidentified but distinct factor. The definition of an octamer-binding protein site within these domains is of interest, as this class of factor has recently been suggested as mediating the effect of nerve growth factor in sensory neurons. Nerve growth factor is a well-characterized regulator of preprotachykinin-A gene expression. Definition of these sites within the promoter allows for the design of rational experiments to address the mechanism of transcriptional regulation of the rat preprotachykinin-A gene.

Chronic testosterone propionate treatment decreases the concentration of [3H]quipazine binding at 5-HT3 receptors in the amygdala of the castrated male rat.

Chronic administration of testosterone propionate (TP) was found to decrease the concentration of [3H]quipazine binding at 5-HT3 receptors in the lateral and basal amygdaloid nuclei of the brains of castrated male rats. TP had no effect on the binding of [3H]quipazine at 5-HT3 receptors in the posterolateral or posteromedial cortical amygdaloid nuclei, or in the amygdalohippocampal areas. It is suggested that the effects of testosterone on sexual and other social behaviors in male rats may be mediated, at least in part, by decreases in the activation of 5-HT3 receptors in the amygdala.

Stress and antidepressant effects on hippocampal and cortical 5-HT1A and 5-HT2 receptors and transport sites for serotonin.

The interactions between 14 days of repeated restraint stress and daily administration of imipramine or tianeptine (2 h before the beginning of stress) were investigated in rats to assess responses of 5-HT2 and 5-HT1A receptors and serotonin transporter sites labelled by [3H]paroxetine in the cerebral cortex and hippocampus, two brain regions in which adrenal steroid effects on serotonin receptor-binding have been reported. 5-HT2 sites, labelled by [125I]7-amino-8-iodo ketanserin, were decreased in parietal cerebral cortex layers 3 and 5 by imipramine treatment, but not by tianeptine treatment and not by daily restraint stress. Stress, but not antidepressant, depressed 5-HT1A sites labelled with [3H]8-hydroxy-DPAT in hippocampal fields CA3, CA4 and dentate gyrus. [3H]paroxetine-binding to serotonin transporter sites was decreased by tianeptine treatment as well as by imipramine in both hippocampus and cerebral cortex, with some overlap of the fields that were significantly affected, whereas there were no effects of stress per se and no evidence of a stress x drug interaction. These results are discussed in relation to similarities and differences in the effects of different antidepressant drugs on the serotonergic system of the rat brain. Whereas the actions of imipramine and tianeptine on 5-HT2 and 5-HT1A receptors are specific to each drug, the surprising finding of a similar effect of both drugs to reduce serotonin transporter sites labelled by [3H]paroxetine suggest the possibility of a common action for these two drugs in spite of their opposite effects on serotonin re-uptake.

Autoradiographic analyses of the effects of estradiol benzoate on [3H]paroxetine binding in the cerebral cortex and dorsal hippocampus of gonadectomized male and female rats.

Effects of estradiol benzoate (EB) on [3H]paroxetine binding in dorsal hippocampus and cerebral cortex of gonadectomized male and female rats were evaluated by quantitative autoradiography. EB significantly decreased [3H]paroxetine binding in male and female rats in the oriens layers of CA1-CA4, and in the radiata/lacunosum moleculare layers of CA2 and CA3. Sex differences were also noted, with binding of [3H]paroxetine being significantly lower in female rats in the radiata/lacunosum moleculare layers of CA2 and CA4, and in the suprapyramidal dentate. No significant effects of either EB or sex were noted in the cortex.

Alterations of serotonin receptor binding in the hypothalamus following acute denervation.

Quantitative autoradiography was used to determine the effect of acute serotonergic denervation with 5,7-dihydroxytryptamine (5,7-DHT) or serotonin 5HT1a and 5-HT1b receptors in male rats. Seven days after intrahypothalamic 5,7-DHT injection there was a significant increase in the density of 5HT1a receptors in the ventromedial and dorsomedial hypothalamic nuclei (VMN and DMN) of male rats. In adjacent sections. 5-HT1b receptors were significantly increased only in the VMN. No changes in receptor density were observed in the lateral hypothalamic area or hippocampus even though binding of [3H]paroxetine, which labels the presynaptic transporter site, was significantly decreased in all evaluated brain regions in 5,7-DHT-treated animals. In addition to demonstrating that 5-HT1a and 5-HT1b receptors are differentially regulated in different brain areas, these results show that in the brain regions examined both 5-HT1a and 5-HT1b receptors are primarily post-synaptic.

Effects of 5-HT1A selective anxiolytics on lordosis behavior: interactions with progesterone.

Ipsapirone and gepirone, but not buspirone, facilitated lordosis in estrogen-treated rats, whereas all three drugs inhibited this behavior in rats treated with estrogen and progesterone. When administered at higher doses, ipsapirone, gepirone and buspirone inhibited lordosis in rats treated with either estrogen or estrogen and progesterone. These data are consistent with the proposal that 5-HT1A receptors mediate lordosis-inhibiting effects of 5-HT, and further suggest that some 5-HT1A agonists may facilitate lordosis by activity at autoreceptors. Finally, these data show that progesterone may modulate activity at 5-HT1A receptors.

Intraventricular administration of l-kynurenine and kynuramine facilitates lordosis in the female rat.

Intraventricular administration of the tryptophan metabolites l-kynurenine (2-32 micrograms) and kynuramine (0.064-8 micrograms) facilitated lordosis behavior in estrogen-primed ovariectomized rats. The facilitatory effects of these drugs were not attenuated by pretreatment with the progesterone antagonist RU 38486, indicating that the effects were not mediated by release of adrenal progesterone. It is suggested that l-kynurenine and kynuramine may serve a physiological role in the modulation of female sexual behavior.