The predictive value of cranial ultrasound and of somatosensory evoked potentials after nerve stimulation for adverse neurological outcome in preterm infants

Thirty-nine preterm infants were studied to compare the predictive value of somatosensory evoked responses (SEPs) following median-nerve and posterior tibial-nerve stimulation with the predictive value of cranial ultrasound. With regard to the SEP, a normal median-nerve response was by no means a guarantee of a normal outcome. A normal posterior tibial-nerve response, however, almost guaranteed a normal outcome, but the test was very time consuming and the number of false positive responses was high (sensitivity. 95.6%, specificity 50%). The presence of parenchymal involvement, either due to a haemorrhage or cystic leukomalacia predicted cerebral palsy with a sensitivity of 95.6% and a specificity of 68.5%. The combination of an abnormal posterior tibial response and the presence of parenchymal brain lesions had the best predictive value with a sensitivity of 91.3% and a specificity of 81.2%. These results show that, although posterior tibial-rferve responses have a better predictive value than median-nerve responses, these values were lower than that of cranial ultrasound. The best prediction was obtained when a combination of posterior-tibial responses and cranial ultrasound was used.     

Nucleotide oligomerization domain 1 is a dominant pathway for NOS2 induction in vascular smooth muscle cells: comparison with Toll-like receptor 4 responses in macrophages

Background and purpose:

Gram-negative bacteria contain ligands for Toll-like receptor (TLR) 4 and nucleotide oligomerization domain (NOD) 1 receptors. Lipopolysaccharide (LPS) activates TLR4, while peptidoglycan products activate NOD1. Activation of NOD1 by the specific agonist FK565 results in a profound vascular dysfunction and experimental shock in vivo.

Experimental approach:

Here, we have analysed a number of pharmacological inhibitors to characterize the role of key signalling pathways in the induction of NOS2 following TLR4 or NOD1 activation.

Key results:

Vascular smooth muscle (VSM) cells expressed NOD1 mRNA and protein, and, after challenge with Escherichia coli or FK565, NOS2 protein and activity were induced. Macrophages had negligible levels of NOD1 and were unaffected by FK565, but responded to E. coli and LPS by releasing increased NO and expression of NOS2 protein. Classic pharmacological inhibitors for NF-κB (SC-514) and mitogen-activated protein kinase (SB203580, PD98059) signalling pathways inhibited responses in both cell types regardless of agonist. While TLR4-mediated responses in macrophages were specifically inhibited by the pan-caspase inhibitor z-VAD-fmk and the PKC inhibitor Gö6976, NOD1-mediated responses in VSM cells were inhibited by the Rip2 inhibitor PP2.

Conclusions and implications:

Our findings suggest a selective role for NOD1 in VSM cells, and highlight NOD1 as a potential novel therapeutic target for the treatment of vascular inflammation.     

The structural basis for the mutagenicity of O(6)-methyl-guanine lesions

Methylating agents are widespread environmental carcinogens that generate a broad spectrum of DNA damage. Methylation at the guanine O(6) position confers the greatest mutagenic and carcinogenic potential. DNA polymerases insert cytosine and thymine with similar efficiency opposite O(6)-methyl-guanine (O6MeG). We combined pre-steady-state kinetic analysis and a series of nine x-ray crystal structures to contrast the reaction pathways of accurate and mutagenic replication of O6MeG in a high-fidelity DNA polymerase from Bacillus stearothermophilus. Polymerases achieve substrate specificity by selecting for nucleotides with shape and hydrogen-bonding patterns that complement a canonical DNA template. Our structures reveal that both thymine and cytosine O6MeG base pairs evade proofreading by mimicking the essential molecular features of canonical substrates. The steric mimicry depends on stabilization of a rare cytosine tautomer in C.O6MeG-polymerase complexes. An unusual electrostatic interaction between O-methyl protons and a thymine carbonyl oxygen helps stabilize T.O6MeG pairs bound to DNA polymerase. Because DNA methylators constitute an important class of chemotherapeutic agents, the molecular mechanisms of replication of these DNA lesions are important for our understanding of both the genesis and treatment of cancer.     

Structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis

Even though high-fidelity polymerases copy DNA with remarkable accuracy, some base-pair mismatches are incorporated at low frequency, leading to spontaneous mutagenesis. Using high-resolution X-ray crystallographic analysis of a DNA polymerase that catalyzes replication in crystals, we observe that a C•A mismatch can mimic the shape of cognate base pairs at the site of incorporation. This shape mimicry enables the mismatch to evade the error detection mechanisms of the polymerase, which would normally either prevent mismatch incorporation or promote its nucleolytic excision. Movement of a single proton on one of the mismatched bases alters the hydrogen-bonding pattern such that a base pair forms with an overall shape that is virtually indistinguishable from a canonical, Watson-Crick base pair in double-stranded DNA. These observations provide structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis, a long-standing concept that has been difficult to demonstrate directly.     

Prevalence of Murine Helicobacter spp. Infection Is Reduced by Restocking Research Colonies with Helicobacter-Free Mice

Most academic research colonies of mice are endemically infected with enterohepatic Helicobacter spp. (EHS). We evaluated EHS prevalence in surveillance mice before and after a 10-y period of requiring that imported mice be free of EHS by embryo transfer rederivation or purchase from approved vendors. In 2009, composite fecal samples from CD1 surveillance mice representing colony health in 57 rooms located in 6 facilities were evaluated for EHS infection by using PCR assays. Fecal samples were screened with primers designed to detect all known EHS, and positive samples were further assayed by using primers specific for H. hepaticus, H. bilis, H. rodentium, and H. typhlonicus. Most EHS were detected in surveillance mice within the first month of dirty bedding exposure, with prevalence ranging from 0% to 64% as monoinfections or, more commonly, infections with multiple EHS. Compared with 1999 prevalence data, EHS remained endemic in colonies importing the lowest number of EHS-free mice. EHS were absent or the prevalence was greatly reduced in colonies receiving the highest percentage of EHS-free mice. This study demonstrates that the management decision to require exclusive importation of EHS-free mice reduced EHS prevalence on an institutional scale without intensive labor and expense associated with other techniques or interference with research objectives.Abbreviation: EHS, enterohepatic Helicobacter spp.; ET, embryo transfer; Hb, H. bilis; Hh, H. hepaticus; Hm, H. mastomyrinus; Hr, H. rodentium; Ht, H. typhlonicusEnterohepatic Helicobacter spp. (EHS) infections are endemic in the majority of research mouse colonies. In 2007, 84% of mice shipped from academic institutions worldwide for embryo transfer (ET) rederivation at our institution were PCR-positive for EHS. H. hepaticus (Hh) was detected in 64% of the mouse shipments either as a monoinfection or in combination with other EHS including H. bilis (Hb), H. rodentium (Hr), H. typhlonicus (Ht), and H. mastomyrinus (Hm).³⁰ Although EHS generally cause subclinical infection in immunocompetent mice, opportunistic infections have the potential to confound experimental data in mouse models.^9,17,34 Importantly, chronic EHS infection in immunodeficient and select inbred strains of mice can induce liver¹⁰ and lower bowel carcinoma,¹³ typhlocolitis, and rectal prolapse,^16,21,28 and reduce reproductive performance.²⁵ In addition, EHS-induced inflammatory responses may alter host immune responses to unrelated experimental infections (for example, promoting elevated systemic IFNγ responses).^3,20Key challenges to eradication of EHS from rodent colonies are determining infection status, eliminating endemic infections, and instituting management practices that prevent reinfection. EHS are disseminated through fecal–oral transmission within a colony and are transmissible to surveillance mice through dirty-bedding exposure.^1,19,24,32 For routine surveillance, PCR assay of feces or cecal mucosal scrapings for genus-specific Helicobacter 16S rRNA genes is the most efficient means of detecting EHS infection, with speciation (if desired) of positive results by culture, restriction fragment length polymorphism analysis, species-specific PCR, or sequence analysis.³⁴ In 1999, as determined by species-specific PCR assays of cecal scrapings from 59 surveillance mice exposed to dirty bedding from colony mice in 26 rooms representing 4 mouse facilities, EHS were endemic on our campus, with prevalence in surveillance mice of 41% for Hh, 82% for Hr, and 6% for Hb.³² Husbandry practices used to minimize cage-to-cage transmission of EHS included microisolation caging, sanitized forceps to transfer mice, and a cage change order from known Helicobacter-free mice to mice of unknown or known EHS infection status (that is, clean to dirty traffic flow of personnel and equipment).³² Although EHS eradication potentially could be accomplished campus-wide by using labor-intensive antibiotics^7,15 and cross-fostering,^4,29,31 we hypothesized that a more cost-effective approach, without confounding experimental data, would be to restrict importation of mice to EHS-free sources. Vendors were screened to establish that production colonies were SPF for EHS, and a new requirement was instituted for embryo transfer (ET) rederivation of mice obtained from random sources, typically other academic institutions, replacing traditional quarantine practices. This study used PCR data from 1999 and 2009 to evaluate the success of this approach, which was defined as a marked decrease in the prevalence of EHS infection over time.