Infections involving Staphylococcus aureus are often more severe and difficult to treat when the organism assumes a biofilm mode of growth. The polysaccharide poly-N-acetylglucosamine (PNAG), also known as polysaccharide intercellular adhesin, is synthesized by the products of the intercellular adhesin (ica) locus and plays a key role in biofilm formation. Numerous conditions and exogenous factors influence ica transcription and PNAG synthesis, but the regulatory factors and pathways through which these environmental stimuli act have been only partially characterized. We developed a DNA affinity chromatography system to purify potential regulatory proteins that bind to the ica promoter region. Using this technique, we isolated four proteins, including the staphylococcal gene regulator SarA, a MarR family transcriptional regulator of the teicoplaninassociated locus TcaR, DNA-binding protein II, and topoisomerase IV, that bound to the ica promoter. Site-directed deletion mutagenesis of tcaR indicated that TcaR was a negative regulator of ica transcription, but deletion of tcaR alone did not induce any changes in PNAG production or in adherence to polystyrene. We also investigated the role of IcaR, encoded within the ica locus but divergently transcribed from the biosynthetic genes. As has been shown previously in Staphylococcus epidermidis, we found that IcaR was also a negative regulator of ica transcription in S. aureus. We also demonstrate that mutation of icaR augmented PNAG production and adherence to polystyrene. Transcription of the ica locus, PNAG production, and adherence to polystyrene were further increased in a tcaR icaR double mutant. In summary, TcaR appeared to be a weak negative regulator of transcription of the ica locus, whereas IcaR was a strong negative regulator, and in their absence PNAG production and biofilm formation were enhanced.
Poly-N-acetyl-glucosamine (PNAG) is a staphylococcal surface polysaccharide influencing biofilm formation that is also under investigation for its vaccine potential. Antibodies that bind to PNAG with either low (<15%) or high (>90%) levels of acetate are superior at opsonic and protective activity compared with antibodies that bind to PNAG with only high levels (>70%) of acetate. PNAG is synthesized by four proteins encoded within the intercellular adhesin (ica) locus icaADBC. In Staphylococcus epidermidis, icaB encodes a deacetylase needed for the surface retention of PNAG and optimal biofilm formation. In this study, we confirmed that icaB plays a similar role in Staphylococcus aureus and found that an icaB mutant of S. aureus expressed significantly less surface-associated PNAG, was highly susceptible to antibody-independent opsonic killing that could not be enhanced with antibody raised against deacetylated PNAG (dPNAG), and had reduced survival capacity in a murine model of bacteremia. In contrast, an icaB-overexpressing strain produced primarily surface-associated PNAG, was more susceptible to opsonophagocytosis with antibody to dPNAG, and had increased survival in a murine bacteremia model. The highly acetylated secreted PNAG was more effective at blocking opsonic killing mediated by a human monoclonal antibody (mAb) to native PNAG than it was at blocking killing mediated by a human mAb to dPNAG, which by itself was a more effective opsonin. Retention of dPNAG on the surface of S. aureus is key to increased survival during bacteremia and also provides a molecular mechanism explaining the superior opsonic and protective activity of antibody to dPNAG.Staphylococcus aureus and coagulase-negative staphylococci are the most frequent causes of nosocomial bloodstream infections (43). A critical virulence determinant in such infections is the production of a high-molecular-weight polymer of -1-6-linked N-acetyl-glucosamine (PNAG) that is involved in adherence to polymeric substrates, bacterial intercellular adhesion, biofilm formation, and protection against antibodyindependent opsonic killing (3,4,15,26,40). Proteins encoded by the icaADBC genes of the intercellular adhesin (ica) locus synthesize PNAG (6, 10-12, 30, 31). IcaA is a trans-membrane glucosyltransferase and can synthesize short PNAG polymers in vitro using UDP-N-acetyl-glucosamine as a substrate (10). IcaD increases the biosynthetic efficiency of IcaA (10). IcaC is also a transmembrane protein and appears to be involved in linking short polymers to make longer oligomers of PNAG (10). In its mature form, PNAG, also referred to by some researchers as polysaccharide intercellular adhesin (PIA) (12,44), is a mixture of polymers such that about 10 to 20% of the amino groups are not acetylated, which could give some of the polymers within the polysaccharide complex a net positive charge (16,27). It is not known whether PNAG consists of a minority of highly deacetylated molecules mixed in with a majority of highly acetylated molecules or if there is a rang...
Objective: To determine brain magnetic resonance imaging (MRI) measures of cerebrospinal fluid (CSF) and whole brain volume of full-term and premature infants following surgical treatment for thoracic non-cardiac congenital anomalies requiring critical care. Methods: Full-term ( n = 13) and pre-term ( n = 13) patients with long-gap esophageal atresia, and full-term naïve controls ( n = 19) < 1 year corrected age, underwent non-sedated brain MRI following completion of thoracic non-cardiac surgery and critical care treatment. Qualitative MRI findings were reviewed and reported by a pediatric neuroradiologist and neurologist. Several linear brain metrics were measured using structural T1-weighted images, while T2-weighted images were required for segmentation of total CSF and whole brain tissue using the M orphologically A daptive N eonatal T issue S egmentation ( MANTiS ) tool. Group differences in absolute (mm, cm 3 ) and normalized (%) data were analyzed using a univariate general linear model with age at scan as a covariate. Mean normalized values were assessed using one-way ANOVA. Results: Qualitative brain findings suggest brain atrophy in both full-term and pre-term patients. Both linear and volumetric MRI analyses confirmed significantly greater total CSF and extra-axial space, and decreased whole brain size in both full-term and pre-term patients compared to naïve controls. Although linear analysis suggests greater ventricular volumes in all patients, volumetric analysis showed that normalized ventricular volumes were higher only in premature patients compared to controls. Discussion: Linear brain metrics paralleled volumetric MRI analysis of total CSF and extra-axial space, but not ventricular size. Full-term infants appear to demonstrate similar brain vulnerability in the context of life-saving thoracic non-cardiac surgery requiring critical care as premature infants.
The hereditary spastic paraplegias (HSPs) are a heterogeneous group of disorders characterized by degeneration of the corticospinal and spinocerebellar tracts leading to progressive spasticity. One subtype, spastic paraplegia type 47 (SPG47 or HSP-AP4B1), is due to bi-allelic loss-of-function mutations in the AP4B1 gene. AP4B1 is a subunit of the adapter protein complex 4 (AP-4), a heterotetrameric protein complex that regulates the transport of membrane proteins. Since 2011, 11 individuals from six families with AP4B1 mutations have been reported, nine of whom had homozygous mutations and were from consanguineous families. Here we report eight patients with AP4B1-associated SPG47, the majority born to non-consanguineous parents and carrying compound heterozygous mutations. Core clinical features in this cohort and previously published patients include neonatal hypotonia that progresses to spasticity, early onset developmental delay with prominent motor delay and severely impaired or absent speech development, episodes of stereotypic laughter, seizures including frequent febrile seizures, thinning of the corpus callosum, and delayed myelination/white matter loss. Given that some of the features of AP-4 deficiency overlap with those of cerebral palsy, and the discovery of the disorder in non-consanguineous populations, we believe that AP-4 deficiency may be more common than previously appreciated.
Objectives To assess the frequency and cause of variability in diagnosis on cranial sonography and magnetic resonance imaging (MRI)
Objective To correlate MR 2D measurements of lateral ventricular width and 3D measures of lateral ventricular and supratentorial parenchymal volumes to postnatal outcomes in fetuses with ventriculomegaly (VM). Methods 307 fetuses (mean gestational age 26.0 weeks, range 15.7-39.4 weeks) had MR volumetry after referral for VM. Fetuses were grouped into those with (N=114) or without (N=193) other CNS anomalies. Pregnancy outcome and postnatal neurodevelopmental outcomes up to age 3 were obtained. A subgroup analysis was performed excluding fetuses with other CNS anomalies. Logistic regression analysis was performed to assess which measure was most predictive of outcome. Results There were 50 terminations and 2 stillbirths. There were 255 live births. 75 were lost to follow-up. Among 180 liveborn infants with follow-up, 140 had an abnormal and 40 had normal outcome. Atrial diameter (p<0.0001), frontal horn diameter (p<0.0001), and ventricular volume (p=0.04) were each predictive of live-birth, with each having 92% specificity at 60% sensitivity. Among fetuses without other CNS anomalies, 180/193 (93%) pregnancies resulted in live deliveries, with atrial diameter (p<0.0001), frontal horn diameter (p=0.003), and ventricular volume (p=0.008) associated with live birth, and with atrial diameter having highest specificity of >99% at 60% sensitivity. Parenchymal volume was not associated with normal or abnormal outcome (either livebirth vs. demise or normal vs. abnormal neurodevelopmental outcome). Among live-borns, there was no age-adjusted threshold for any of the measures that reliably distinguished between normal and abnormal neurodevelopmental outcome. Conclusions Ventricular volume and diameter, but not parenchymal volume, correlate with live birth in fetuses with VM. However, once live-born, neither 2D nor 3D measurements can distinguish a fetus that will go on to have a normal outcome.
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