Suppressor analysis links trans-translation and ribosomal protein uS7 to RluD function in Escherichia coli.

The pseudouridine (ψ) synthase, RluD is responsible for three ψ modifications in the helix 69 (H69) of bacterial 23S rRNA. While normally dispensable, rluD becomes critical for rapid cell growth in bacteria that are defective in translation-termination. In slow-growing rluD- bacteria, suppressors affecting termination factors RF2 and RF3 arise frequently and restore normal termination and rapid cell growth. Here we describe two weaker suppressors, affecting rpsG, encoding ribosomal protein uS7 and ssrA, encoding tmRNA. In K-12 strains of E. coli, rpsG terminates at a TGA codon. In the suppressor strain, alteration of an upstream CAG to a TAG stop codon results in a shortened uS7 and partial alleviation of slow growth, likely by replacing an inefficient TGA stop codon with the more efficient TAG. Inefficient termination events, such as occurs in some rluD- strains, are targeted by trans-translation. Inactivation of the ssrA gene in slow-growing, termination-defective mutants lacking RluD and RF3, also partially restores robust growth, most probably by preventing destruction of completed polypeptides on ribosomes at slow-terminating stop codons. Finally, an additional role for RluD has been proposed, independent of its pseudouridine synthase activity. This is based on the observation that plasmids expressing catalytically dead (D139N or D139T) RluD proteins could nonetheless restore robust growth to an E. coli K-12 rluD- mutant. However, newly constructed D139N and D139T rluD plasmids do not have any growth-restoring activity and the original observations were likely due to the appearance of suppressors.

Quantitative 1H-NMR analysis reveals steric and electronic effects on the substrate specificity of benzoate dioxygenase in Ralstonia eutropha B9.

The cis-dihydroxylation of arenes by Rieske dearomatizing dioxygenases (RDDs) represents a powerful tool for the production of chiral precursors in organic synthesis. Here, the substrate specificity of the RDD benzoate dioxygenase (BZDO) in Ralstonia eutropha B9 whole cells was explored using quantitative 1H nuclear magnetic resonance spectroscopy (q1H-NMR). The specific activity, specific carbon uptake, and regioselectivity of the dihydroxylation reaction were evaluated in resting cell cultures for a panel of 17 monosubstituted benzoates. Two new substrates of this dioxygenase system were identified (2-methyl- and 3-methoxybenzoic acid) and the corresponding cis-diol metabolites were characterized. Higher activities were observed for benzoates with smaller substituents, predominantly at the 3-position. Elevated activities were also observed in substrates bearing greater partial charge at the C-2 position of the benzoate ring. The regioselectivity of the reaction was directly measured using q1H-NMR and found to have positive correlation with increasing substituent size. These results widen the pool of cis-diol metabolites available for synthetic applications and offer a window into the substrate traits that govern specificity for BZDO.

Comparisons of trunk and knee mechanics during various speeds of treadmill running between runners with and without patellofemoral pain: a preliminary study.

[Purpose] To determine if runners with patellofemoral pain (PFP) exhibit higher patellofemoral joint (PFJ) stress and trunk extension compared to pain-free runners during treadmill running. [Participants and Methods] Twelve runners (7 with PFP and 5 pain-free) participated in this study. Participants ran at 3 different running conditions: self-selected, fast (120% of self-selected), and slow (80% of self-selected) speeds. Kinematics and kinetics of trunk and lower extremities were obtained. PFJ stress, PFJ reaction force, and PFJ contact area were determined using a biomechanical model. Two-factor ANOVAs with repeated measures were used to compare outcome variables between 3 speeds and between 2 groups. [Results] There was no significant difference in peak PFJ stress between groups across the 3 speeds. Peak PFJ stress was lowest during slow running compared to fast and self-selected running speed conditions across both groups. No significant difference was found in trunk flexion angle, PFJ reaction force, or PFJ contact area between groups across the 3 speeds. [Conclusion] Runners with and without PFP exhibited similar peak PFJ stress and trunk flexion angle during treadmill running. This preliminary work does not support the theory that reduced trunk flexion during running contributes to increased PFJ stress in runners with PFP.

Abdominal rapid-kVp-switching dual-energy MDCT with reduced IV contrast compared to conventional MDCT with standard weight-based IV contrast: an intra-patient comparison.

PURPOSE: Perform intra-patient comparison of attenuation values on lower keV dual-energy abdominal CT images using reduced IV contrast dose compared to conventional single energy polychromatic beam abdominal MDCT images using standard IV contrast dose. METHODS: IRB approved retrospective evaluation of consecutive adults who had both standard IV contrast dosage conventional multiphasic MDCT (SECT) and reduced IV contrast dosage rapid kV-switching dual-energy multiphasic MDCT (rsDECT) of the abdomen. Arterial phase dual-energy 52, 70 and 78 keV simulated monoenergetic HU were compared (t test) to arterial phase SECT HU for: aorta, liver, pancreas, psoas, and hepatic/pancreatic tumors. Contrast to noise ratios (CNR), IV contrast dose reduction and dose-length product (DLP) were recorded. Two blinded independent readers evaluated the CT datasets for subjective image quality based on a five point scale. RESULTS: Twenty-nine scan pairs in 24 subjects (13 M, mean age 64, weight 76.7 kg) were evaluated. Mean reduction in IV contrast dose was 37 %. Mean ± SD HU on 52 keV rsDECT vs. SECT were: aorta 534 ± 138 vs. 271 ± 69; liver 88 ± 24 vs. 67 ± 16; pancreas 140 ± 60 vs. 89 ± 40; psoas 63 ± 15 vs. 50 ± 12 (all p < 0.001). Noise was higher for 52 keV compared to SECT (p < 0.001); CNRs were not significantly different. Mean ± SD DLP for rsDECT was 1421 ± 563 and SECT 1335 ± 562 mGy·cm (p = 0.640). For tumor vs. nontumoral parenchyma, mean absolute contrast difference was 58.4 HU on 52 keV, and 29.0 HU on SECT. Nearly all images were rated as good or excellent and there were no statistically significant differences in image quality between the DECT and SECT images. CONCLUSION: Statistically significant gains in vascular and parenchymal enhancement without adverse effect on CNR or lesion contrast were observed in this intra-patient comparison using reduced IV contrast dose rsDECT compared to standard weight-based IV dose conventional SECT.

Vulnerable periods for the mouse mammary gland: comparison of the effects of ethinyl estradiol exposures during two early stages of development.

The mammary gland is responsive to endogenous hormones and environmental chemicals that are estrogen receptor (ER) agonists. The mouse mammary gland offers the opportunity to dissect the most sensitive windows of exposure. 17α-ethinyl estradiol (EE2) is a pharmaceutical ER agonist that often serves as a positive control for estrogen-active chemicals. Here, adult female mice were exposed to EE2 starting either at pregnancy day 7, or on lactational day 1, and exposures continued until the litters were weaned. The pups were therefore exposed during gestation + the juvenile period, or during the juvenile period alone. The morphology of the mammary gland was evaluated in both male and female offspring at two life stages: weaning (postnatal day [PND]21) and at puberty (PND32). Other hormone-sensitive outcomes evaluated included body weight, anogenital index, frequency of open vagina, and weight of the uterus. We found age- and sex-dependent effects of EE2 on these estrogen-responsive endpoints including the morphology of the mammary gland. Importantly, EE2 altered mammary gland morphology even when exposures were limited to the juvenile period. However, the number of endpoints that were affected in animals from the EE2-Juvenile-Only period were fewer, and typically of a lower magnitude, compared to those observed in the EE2-Gest-Juvenile group. Understanding the effects of environmental estrogen exposures during the juvenile period is critical because humans are exposed to estrogenic pollutants throughout life, including in early childhood.

YjbH contributes to Staphylococcus aureus skin pathology and immune response through Agr-mediated α-toxin regulation.

Staphylococcus aureus is the most common cause of skin and soft tissue infections (SSTIs) with Methicillin-Resistant S. aureus (MRSA) strains being a major contributor in both community and hospital settings. S. aureus relies on metabolic diversity and a large repertoire of virulence factors to cause disease. This includes α-hemolysin (Hla), an integral player in tissue damage found in various models, including SSTIs. Previously, we identified a role for the Spx adapter protein, YjbH, in the regulation of several virulence factors and as an inhibitor of pathogenesis in a sepsis model. In this study, we found that YjbH is critical for tissue damage during SSTI, and its absence leads to decreased proinflammatory chemokines and cytokines in the skin. We identified no contribution of YjbI, encoded on the same transcript as YjbH. Using a combination of reporters and quantitative hemolysis assays, we demonstrated that YjbH impacts Hla expression and activity both in vitro and in vivo. Additionally, expression of Hla from a non-native promoter reversed the tissue damage phenotype of the ΔyjbIH mutant. Lastly, we identified reduced Agr activity as the likely cause for reduced Hla production in the ΔyjbH mutant. This work continues to define the importance of YjbH in the pathogenesis of S. aureus infection as well as identify a new pathway important for Hla production.

Effects of butyl benzyl phthalate exposure during pregnancy and lactation on the post-involution mammary gland.

The mammary gland undergoes comprehensive reorganization during pregnancy, lactation, and subsequent involution. Following involution, the mammary gland has structural and functional differences compared to the gland of a nulliparous female. These parity-associated changes are regulated by hormones and may be vulnerable to endocrine-disrupting chemicals (EDCs). In this study, we evaluated the long-term effects of butyl benzyl phthalate (BBP), an estrogenic plasticizer, on the parous mouse mammary gland. Pregnant BALB/c mice were treated with 0, 3, 500, or 18000 µg/kg/day BBP throughout both pregnancy and the lactational period. The litters born to these females were evaluated for litter size and growth. The parous females were then kept for five weeks following weaning of the pups, during which period there was no exposure to BBP. After five weeks of post-weaning, mammary glands were collected and assessed for changes in histomorphology, steroid receptor expression, innate immune cell number, and gene expression. An unexposed age-matched nulliparous control was also evaluated as a comparator group. BBP increased male and female pup weight at puberty and female offspring in adulthood. BBP also altered innate immune cells in the post-involution mammary gland, reducing the effect of parity on macrophages. Lastly, BBP modestly increased mammary gland ductal complexity and periductal structure, but had no effect on expression of estrogen receptor, progesterone receptor, or a marker of proliferation. These results suggest that BBP may interfere with some effects of parity on the mouse mammary gland and induce weight gain in exposed offspring.

Aseptic Viral Meningitis Secondary to Herpes Simplex Virus 2 Genital Infection.

Aseptic viral meningitis is the most common cause of meningitis in the United States. Most cases of herpes simplex virus meningitis are caused by herpes simplex virus type 2 (HSV-2), with HSV-1 primarily causing viral encephalitis. In this report, we present a case of aseptic meningitis generated by the HSV-2 in an immunosuppressed 35-year-old female with a recent diagnosis of genital herpes that was left untreated due to reported side effects of medication.

A Case of Blunt Trauma-induced ST-elevation Myocardial Infarction Sustained During a Prison Brawl.

Myocardial infarction (MI) is a serious and time-sensitive condition. MIs are typically seen in patients with coronary artery disease (CAD) and are caused by the rupture of an atherosclerotic plaque due to factors contributing to plaque instability. However, this case illustrates that plaque rupture can also be caused by blunt trauma to the chest. Considering MI as a possible result of chest trauma may decrease time from presentation to diagnosis and treatment and, therefore, improve outcomes in similar cases, particularly when patients presents unusually or with very few risk factors for MI.

Non contrast, Pseudo-Continuous Arterial Spin Labeling and Accelerated 3-Dimensional Radial Acquisition Intracranial 3-Dimensional Magnetic Resonance Angiography for the Detection and Classification of Intracranial Arteriovenous Shunts.

OBJECTIVES: The aim of this study was to assess the sensitivity and specificity of pseudo-continuous arterial spin labeling (PCASL) magnetic resonance angiography (MRA) with 3-dimensional (3D) radial acquisition for the detection of intracranial arteriovenous (AV) shunts. MATERIALS AND METHODS: A total of 32 patients who underwent PCASL-MRA, clinical magnetic resonance imaging (MRI)/MRA exam, and digital subtraction angiography (DSA) were included in this retrospective analysis. Twelve patients presented with AV shunts. Among these were 8 patients with AV malformations (AVM) and 4 patients with AV fistulas (AVF). The clinical MRI/MRA included 3D time-of-flight MRA in all cases and time-resolved, contrast-enhanced MRA in 9 cases (6 cases with AV shunting). Research MRI and clinical MRI were independently evaluated by 2 neuroradiologists blinded to patient history. A third radiologist evaluated DSA imaging. A diagnostic confidence score was used for the presence of abnormalities associated with AV shunting (1-5). The AVMs were characterized using the Spetzler-Martin scale, whereas AVFs were characterized using the Borden classification. κ Statistics were applied to assess intermodality agreement. RESULTS: Compared with clinical MRA, noncontrast PCASL-MRA with 3D radial acquisition yielded excellent sensitivity and specificity for the detection of intracranial AV shunts (reader 1: 100%/100%, clinical MRA: 91.7%, 94.4%; reader 2: 91.7%/100%, clinical MRA: 91.7%/100%). Diagnostic confidence was 4.8/4.66 with PCASL-MRA and 4.25/4.66 with clinical MRA. For AVM characterization with PCASL-MRA, intermodality agreement with DSA showed κ values of 0.43 and 0.6 for readers 1 and 2, respectively. For AVF characterization, intermodality agreement showed κ values of 0.56 for both readers. CONCLUSION: Noncontrast PCASL-MRA with 3D radial acquisition is a potential tool for the detection and characterization of intracranial AV shunts with a sensitivity and specificity equivalent or higher than routine clinical MRA.

Accelerated Time-Resolved Contrast-Enhanced Magnetic Resonance Angiography of Dural Arteriovenous Fistulas Using Highly Constrained Reconstruction of Sparse Cerebrovascular Data Sets.

OBJECTIVE: Time-resolved contrast-enhanced magnetic resonance angiography (MRA) is commonly used to noninvasively characterize vascular malformations. However, the spatial and temporal resolution of current methods often compromises the clinical value of the examinations. Constrained reconstruction is a temporal spatial correlation strategy that exploits the relative sparsity of vessels in space to dramatically reduce the amount of data required to generate fast high-resolution time-resolved contrast-enhanced MRA studies. In this report, we use a novel temporal spatial acceleration method termed HYPRFlow to diagnose and classify dural arteriovenous fistulas (DAVFs). Our hypothesis is that HYPRFlow images are of adequate diagnostic image quality to delineate the arterial and venous components of DAVFs and allow correct classification using the Cognard system. SUBJECTS AND METHODS: Eight patients with known DAVFs underwent HYPRFlow imaging with isotropic resolution of 0.68 mm and temporal resolution of 0.75 second and 3-dimensional time-of-flight (3DTOF) MRA. The 3DTOF images and HYPRFlow images were evaluated by 2 readers and scored for arterial anatomic image quality. Digital subtraction angiography (DSA) was available for comparison in 7 subjects, and for these patients, each DAVF was classified according to the Cognard system using HYPRFlow and DSA examinations. Digital subtraction angiography was considered the reference examination or criterion standard. RESULTS: HYPRFlow imaging classification was concordant with DSA in all but 1 case. There was no difference in the arterial image quality scores between HYPRFlow and 3DTOF MRA (95% confidence interval). Arterial-to-venous separation was rated excellent (n = 3), good (n = 4), or poor (n = 1), and arteriovenous shunting was easily appreciated. Undersampling artifacts were reduced by using a low pass filter and did not interfere with the diagnostic quality of the examinations. CONCLUSIONS: HYPRFlow is a novel acquisition and reconstruction technique that exploits the relative sparsity of intracranial vessels in space to increase temporal and spatial resolution and provides accurate delineation of DAVF vasculature.

In Vivo application of RNAi to study pain.

Chronic pain is associated with several disease conditions. The inadequacy of current analgesics to treat chronic pain is the result of a lack of understanding of the mechanisms that mediate pain. RNA interference has emerged in recent years as a new way to evaluate the roles of molecules involved in the pain response. Selective knockout of proteins has proven to be a powerful technique for target validation, but has been limited as a potential therapeutic due to short-lived responses induced by RNAi. The short responses of RNAi illustrate the need for better delivery techniques, which is being addressed by current work to induce RNAi through the cell's natural mechanisms. In order to gain a better understanding of chronic pain, it will be necessary to evaluate the pain molecules that are expressed as part of an injury induced pain response, which can be modeled by contusion spinal cord injury. RNAi will prove to be an important technique in this work. The present minireview will summarize the work that has been done using RNAi in vivo to study pain and discuss future directions for the use of RNAi to study chronic pain.