Regulatory roles of RNA binding proteins in the nervous system of C. elegans.
Neurons have evolved to employ many factors involved in the regulation of RNA processing due to their complex cellular compartments. RNA binding proteins (RBPs) are key regulators in transcription, translation, and RNA degradation. Increasing studies have shown that regulatory RNA processing is critical for the establishment, functionality, and maintenance of neural circuits. Recent advances in high-throughput transcriptomics have rapidly expanded our knowledge of the landscape of RNA regulation, but also raised the challenge for mechanistic dissection of the specific roles of RBPs in complex tissues such as the nervous system. The C. elegans genome encodes many RBPs conserved throughout evolution. The rich analytic tools in molecular genetics and simple neural anatomy of C. elegans offer advantages to define functions of genes in vivo at the level of a single cell. Notably, the discovery of microRNAs has had transformative effects to the understanding of neuronal development, circuit plasticity, and neurological diseases. Here we review recent studies unraveling diverse roles of RBPs in the development, function, and plasticity of C. elegans nervous system. We first summarize the general technologies for studying RBPs in C. elegans. We then focus on the roles of several RBPs that control gene- and cell-type specific production of neuronal transcripts.


NeBULA: A Standardized Protocol for the Benchmarking of Robotic-based Upper Limb Neurorehabilitation.
The use of robotic technologies in neurorehabilitation is growing, because they allow highly repeatable exercise protocols and patient-tailored therapies. However, there is a lack of objective methods for assessing these technologies, which makes it difficult to determine their value in rehabilitation settings. While there exist many outcome measurements for motor assessment from a clinical standpoint (such as the Fugl-Meyer scale), the evaluation of performance and clinical benefits of technology for rehabilitation still lacks a standardized approach from a technical standpoint.In this work, we describe NeBULA (Neuromechanical Biomarkers for Upper Limb Assessment), a benchmarking platform for evaluating robotic technology for upper limb neurorehabilitation. By utilizing standardized neuromechanical biomarkers, NeBULA aims at providing a groundwork for assessing and comparing neurorehabilitation robots. We describe its implementation and preliminary results assessing a novel upper limb exoskeleton.Clinical Relevance- Standardized evaluation of neurorehabilitation robots can lead to better patient outcomes, optimizing resources by identifying the most effective technology and by boosting their use in clinical practice. This would provide quantitative and objective information to complement clinical motor evaluation - preventing suboptimal treatments and ensuring that patients receive personalized care. It can also facilitate the transfer of technologyto clinics, identifying the most promising ones for further investment and research.


How does a skilled nursing facility get reimbursed by Medicare? Interview by Kathleen D. Schaum.
Skilled nursing facility wound care nurses should do their part to thoroughly and accurately complete the MDS for each patient. They should remember to manage the wounds aggressively from day 1. They should also remember to consider the total cost of care rather than the cost of a dressing, drug, or piece of equipment. These wound care nurses should coordinate care with wound care physicians and HOPDs when patients require their services. Finally, SNF wound care nurses should take time to read and learn the surgical dressing and negative-pressure LCDs to ensure that they obtain the proper orders and that they follow the utilization and documentation guidelines required for Medicare Part B coverage of these products.


Effect of soybean meal origin on standardized ileal amino acid digestibility in piglets.
The present study with piglets was conducted to estimate standardized ileal digestibility (SID) of AA in European Union soybean (Glycine max) meal (SBM) imports sourced from Argentina, Brazil, and the United States. Twelve piglets with an average initial BW of 9 kg (47 d of age) were fitted with a simple ileal T-cannula to determine SID of AA in 4 batches of SBM originating either from Argentina (n = 1), Brazil (n = 1), or the United States (n = 2; US1 and US2). A semisynthetic diet based on corn (Zea mays) starch and casein (125 g casein/kg as-fed) was supplemented with 1 of 4 batches SBM at an inclusion rate of 235 g SBM/kg (as-fed) each. The chemical composition did not differ largely, with CP contents ranging from 458 in Argentinean to 461 g/kg (as-fed) in US1 SBM. The SID of indispensable AA ranged from 80% in US2 SBM to 83% in Argentinean SBM and from 74% for Trp in US2 and Brazilian SBM to 91% for Arg in US2, Brazilian, and Argentinean SBM. Most SID values did not differ among the SBM batches (P > 0.05). In conclusion, European SBM imports from Brazil, Argentina, or the United Sates were similar in their chemical composition. For most AA, high and uniform SID values were obtained independent from their source of origin.


Assessment of an audio-visual feedback device used in motor training,.
A miniature biofeedback device, the Basmajian-Emory Muscle Trainer, has been developed at the Emory University School of Medicine. A training and testing protocol was designed for using this device in the training of precise voluntary control of individual skeletal muscles. Thirteen normal subjects were trained to abduct the big toe of the right foot, eleven of whom displayed increases in maximum motion. Use of a biofeedback device to improve a subject's ROM capabilities requires consideration of four factors: (1) initial emphasis must be on making the subject aware of his muscle activity, (2) the muscle activity must be increased, (3) the positive re-enforcement of feedback must be continual, and (4) the newly acquired muscle activity must be used to perform a greattting.


Cell fusion by nasopharyngeal carcinoma-derived Epstein-Barr virus.
Cell fusion experiments using Epstein-Barr virus (EBV) obtained from nasopharyngeal carcinoma (NPC) hybrid cells (NPC-KT) were carried out to examine whether human adenoid epithelial cells (Ad-AH) could be fused with lymphoblastoid cells (BJAB, Raji, and A2L) superinfected or infected by EBV, and if Ad-AH nuclei of the fused cells could express EBV-associated nuclear antigen. The presence of NPC EBV could induce EBV early antigen in all three lymphoblastoid cell lines. By autoradiography, we found that NPC EBV could induce cell fusion between Ad-AH cells and tritiated thymidine-labeled lymphoblastoid cells. In addition, we found that Ad-AH nuclei of the fused cells expressed EBV-associated nuclear antigen four days after NPC EBV-mediated cell fusion of Ad-AH cells with lymphoblastoid cells.


Endovascular vs. Open Repair for Ruptured Abdominal Aortic Aneurysm.
Patients presenting with ruptured abdominal aortic aneurysms are most often treated with open repair despite the fact that endovascular aneurysm repair is a less invasive and widely accepted method with clear benefits for elective aortic aneurysm patients. A debate exists regarding the definitive benefit in endovascular repair for patients with a ruptured abdominal aortic aneurysm. The aim of this literature review was to determine if any trends exist in favor of either open or endovascular repair. A literature search was performed using PUBMED, OVID, and Google Scholar databases. The search yielded 64 publications. Out of 64 publications, 25 were retrospective studies, 12 were population-based, 21 were prospective, 5 were the results of RCTs, and 1 was a case-series. Sixty-one studies reported on early mortality and provided data comparing endovascular repair (rEVAR) and open repair (rOR) for ruptured abdominal aneurysm groups. Twenty-nine of these studies reported that rEVAR has a lower early mortality rate. Late mortality after rEVAR compared to that of rOR was reported in 21 studies for a period of 3 to 60 months. Results of 61.9% of the studies found no difference in late mortality rates between these 2 groups. Thirty-nine publications reported on the incidence of complications. Approximately half of these publications support that the rEVAR group has a lower complication rate and the other half found no difference between the groups. Length of hospital stay has been reported to be shorter for rEVAR in most studies. Blood loss and need for transfusion of either red cells or fresh frozen plasma was consistently lower in the rEVAR group. Differences between the included publications affect the outcomes. Randomized control trials have not been able to provide clear conclusions. rEVAR can now be considered a safe method of treating rAAA, and is at least equal to the well-established rOR method.


Measuring Opioid Withdrawal in a Phase 3 Study of a New Analgesic, NKTR-181 (Oxycodegol), in Patients with Moderate to Severe Chronic Low Back Pain.
To evaluate the SUMMIT-07 trial opioid withdrawal results of NKTR-181 (oxycodegol), a new molecular entity mu-opioid receptor agonist. Phase 3, enriched-enrollment, double-blind, randomized-withdrawal study in patients with chronic low back pain (CLBP). Conducted in the United States at multiple sites. SUMMIT-07 was comprised of five periods: screening; NKTR-181 open-label titration (100 to 400 mg twice daily); 12-week randomized, double-blind study drug (NKTR-181 or placebo); one-week study drug taper; and two-week safety follow-up. Permitted rescue medication included hydrocodone 5 mg/acetaminophen 300 mg (two tablets daily) for two weeks after randomization, then acetaminophen 1.0 gm daily for the remainder of the trial. Signs and symptoms of drug withdrawal were evaluated using the Clinical Opiate Withdrawal Scale (COWS); Subjective Opiate Withdrawal Scale (SOWS); Misuse, Abuse, and Diversion Drug Event Reporting System (MADDERS); and withdrawal-related adverse events. Of 1,190 patients entering titration, one patient had moderate withdrawal (COWS score 13/48 maximum) three days after discontinuing NKTR-181. Of 610 patients randomized (N = 309, NKTR-181; N = 301, placebo), no COWS scores indicating withdrawal at a moderate level or greater (i.e., score ≥13) were observed at any time point. At day 8 after randomization, week 12, and the end of tapering, COWS scores indicating mild withdrawal (<13) were observed in seven (2.4%), one (0.4%), and one (0.5%) placebo patients, respectively, and three (1.0%), one (0.4%), and five (2.3%) NKTR-181 patients, respectively. Mean SOWS scores in both arms were ≤2.8 of 64 possible points at all time points. During the randomized period, of 35 events identified by MADDERS, adjudicators identified 20 possible "withdrawal" events (9 [2.9%] NKTR-181 and 11 [3.7%] placebo). NKTR-181 exhibited a low rate and severity of opioid withdrawal in SUMMIT-07 patients with CLBP.


Comprehensive Influences of Overexpression of a MYB Transcriptor Regulating Anthocyanin Biosynthesis on Transcriptome and Metabolome of Tobacco Leaves.
Overexpression of R2R3-MYB transcriptor can induce up-expression of anthocyanin biosynthesis structural genes, and improve the anthocyanin content in plant tissues, but it is not clear whether the MYB transcription factor overexpression does effect on other genes transcript and chemical compounds accumulation. In this manuscript, RNA-sequencing and the stepwise multiple ion monitoring-enhanced product ions (stepwise MIM-EPI) strategy were employed to evaluate the comprehensive effect of the MYB transcription factor LrAN2 in tobacco. Overexpression of LrAN2 could promote anthocyanin accumulation in a lot of tissues of tobacco cultivar Samsun. Only 185 unigenes express differently in a total of 160,965 unigenes in leaves, and 224 chemical compounds were differently accumulated. Three anthocyanins, apigeninidin chloride, pelargonidin 3-O-beta-D-glucoside and cyanidin 3,5-O-diglucoside, were detected only in transgenic lines, which could explain the phenotype of purple leaves. Except for anthocyanins, the phenylpropanoid, polyphenol (catechin), flavonoid, flavone and flavonol, belong to the same subgroups of flavonoids biosynthesis pathway with anthocyanin and were also up-accumulated. Overexpression of LrAN2 activated the bHLH (basic helix-loop-helix protein) transcription factor AN1b, relative to anthocyanin biosynthesis and the MYB transcription factor MYB3, relative to proanthocyanin biosynthesis. Then, the structural genes, relative to the phenylpropanoid pathway, were activated, which led to the up-accumulation of phenylpropanoid, polyphenol (catechin), flavonoid, flavone, flavonol and anthocyanin. The MYB transcription factor CPC, negative to anthocyanin biosynthesis, also induced up-expression in transgenic lines, which implied that a negative regulation mechanism existed in the anthocyanin biosynthesis pathway. The relative contents of all 19 differently accumulated amino and derivers were decreased in transgenic lines, which meant the phenylalanine biosynthesis pathway completed the same substrates with other amino acids. Interestingly, the acetylalkylglycerol acetylhydrolase was down-expressed in transgenic lines, which caused 19 lyso-phosphatidylcholine and derivatives of lipids to be up-accumulated, and 8 octodecane and derivatives were down-accumulated. This research will give more information about the function of MYB transcription factors on the anthocyanin biosynthesis and other chemical compounds and be of benefit to obtaining new plant cultivars with high anthocyanin content by biotechnology.


High-purity isolation of bullfrog hair bundles and subcellular and topological localization of constituent proteins.
The small number of hair cells in auditory and vestibular organs severely impedes the biochemical characterization of the proteins involved in mechano-electrical transduction. By developing an efficient and clean "twist-off" method of hair bundle isolation, and by devising a sensitive, nonradioactive method to detect minute quantities of protein, we have partially overcome this limitation and have extensively classified the proteins of the bundles. To isolate hair bundles, we glue the saccular macula of the bullfrog to a glass coverslip, expose the tissue to a molten agarose solution, and allow the agarose to solidify to a firm gel. By rotating the gel disk with respect to the fixed macula, we isolate the hair bundles by shearing them at their mechanically weak bases. The plasma membranes of at least 80% of the stereocilia reseal. To visualize the proteins of the hair bundle, we covalently label them with biotin, separate them by SDS-PAGE, and transfer them to a charged nylon membrane. We can detect less than 500 fg of protein by probing the membrane with streptavidin-alkaline phosphatase and detecting the chemiluminescent product from the hydrolysis of the substrate 3-(4-methoxyspiro-(1,2-dioxetane-3,2'-tricyclo-[3.3.1. 1(3.7)]decan)-4-yl) phenyl phosphate (AMPPD). These techniques reveal a distinct constellation of proteins in and associated with hair bundles. Several proteins, such as calmodulin, calbindin, actin, tubulin, and fimbrin, have previously been described. A second class of proteins in the preparation appears to be derived from extracellular sources. Finally, several heretofore undescribed bundle proteins are identified and characterized by their membrane topology, subcellular localization, and glycosidase and protease sensitivities.


Essentialism, word use, and concepts.
The essentialist approach to word meaning has been used to undermine the fundamental assumptions of the cognitive psychology of concepts. Essentialism assumes that a word refers to a natural kind category in virtue of category members possessing essential properties. In support of this thesis, Kripke and Putnam deploy various intuitions concerning word use under circumstances in which discoveries about natural kinds are made. Although some studies employing counterfactual discoveries and related transformations appear to vindicate essentialism, we argue that the intuitions have not been investigated exhaustively. In particular, we argue that discoveries concerning the essential properties of whole categories (rather than simply of particular category members) are critical to the essentialist intuitions. The studies reported here examine such discovery contexts, and demonstrate that words and concepts are not used in accordance with essentialism. The results are, however, consistent with "representational change" views of concepts, which are broadly Fregean in their motivation. We conclude that since essentialism is not vindicated by ordinary word use, it fails to undermine the cognitive psychology of concepts.


[A simple method for the demonstration of the bacterial spectrum in the nose and the nasopharynx in the infection-free interval in children with adenoid hypertrophy].
The implications of bacterial colonization and distribution patterns in the nasopharynx and nasal cavities of children with adenoidal hypertrophy without clinical signs of acute infection are to be determined. We examined the spectrum and distribution of the facultative pathogenic bacterial flora in nasal cavities and nasopharynx of children with clinical apparent symptoms or signs of adenoid hypertrophy in an infection free interval. Compared with the nasal cavity we found an accumulation of pathogenic bacteria in the nasopharynx. A transnasal single swab from the nasopharynx showed to be the most effectively practical way to detect clinically relevant pathogenic bacteria. A thin flexible calcium-alginate swab was used in our experiments. Swabbing from the anterior nasal cavities proved to be a minor successful diagnostic method.


Prediction of Boston Naming Test performance from vocabulary scores: preliminary guidelines for interpretation.
Patients with limited education or underdeveloped vocabulary skills may perform below the normal range on the Boston Naming Test when compared to the original published norms, even in the absence of brain damage. To reduce the frequency of false positive dysnomic classifications of patients with limited vocabulary skills, we developed a score adjustment to account for the significant shared variance between scores on this test and the WAIS-R Vocabulary subtest. Vocabulary significantly predicted performance on the Boston Naming Test (r = .65, p < .0001) in a sample of 62 outpatients who had no objective evidence of brain damage. Linear regression was used to derive expected performance on the Boston Naming Test from Vocabulary scaled scores. Relative to the original published norms, scores based on the Vocabulary subtest cut-offs produced fewer false positives and more accurately classified group membership for patients with and without objectively verified brain damage. These performance predictions are offered as tentative guidelines to assist clinicians in evaluating the presence of naming deficits by controlling for the variance associated with knowledge of vocabulary.


Tumor oxygenation after hyperthermia in the rat 13762 mammary carcinoma and the DU-145 human prostate carcinoma.
Tumor oxygenation was measured in the rat 13762 mammary carcinoma over a time course from 30 min to 3 days after hyperthermia treatment of 43 degrees C, 30 min or 40 degrees C, 80 min, while the animals breathed air, carbogen or after administration of a perflubron emulsion with air or carbogen breathing. Shortly (30 min and 3 h) after treatment with 43 degrees C, 30 min, the 13762 tumors were more hypoxic, as determined by the percent of pO(2) readings <5 mmHg and median pO(2), than prior to treatment and by 24 h had returned to initial oxygenation. Administration of the perflubron emulsion and carbogen breathing abrogated the increase in hypoxia and improved tumor oxygenation. Athymic mice bearing the DU-145 human prostate carcinoma were treated with hyperthermia regimens 40 degrees C, 41 degrees C, 42 degrees C or 43 degrees C for 60 min and tumor oxygenation was then measured over a time course while the animals breathed air or carbogen. Shortly (30 min) after hyperthermia, the DU-145 tumors were more hypoxic than initially when the animals breathed air. Three days later, the DU-145 tumors treated with 41 degrees C: 42 degrees C or 43 degrees C remained hypoxic but the tumors treated with 40 degrees C were better oxygenated than initially. When the animals bearing the DU-145 tumor breathed carbogen during the tumor oxygenation measurements only tumors treated with 43 degrees C were more hypoxic shortly after hyperthermia treatment. Three days after hyperthermia, the DU-145 tumors treated with 40 degrees C and 43 degrees C were better oxygenated than initially while those treated with 41 degrees C and 42 degrees C were less oxygenated when the animals breathed carbogen during the oxygen measurements. Thus, the effect of hyperthermia on tumor physiology can persist for days post treatment and vary with the temperature of the treatment.


Gap junctional coupling in lenses lacking alpha3 connexin.
Fiber cells of the lens are interconnected by an extensive network of gap junctions containing alpha3 (Cx46) and alpha8 (Cx50) connexins. A specific role for these connexins in lens homeostasis is not known. To determine the contribution of these connexins to lens function, we used impedance techniques to study cell-to-cell coupling in lenses from homozygous alpha3 knockout (-/-), heterozygous (+/-), and wild-type (+/+) mice. Western blots and immunofluorescence data indicated that alpha8 remained at similar levels in the three classes of lenses, whereas alpha3 was approximately 50% of the normal level in the +/- lenses, and it was absent from the -/- lenses. Moreover, the data from +/+ lenses suggest that a cleavage of connexins occurs abruptly between the peripheral shell of differentiating fibers (DF) and the inner core of mature fibers (MF). The appearance of the cleaved connexins was correlated to a change in the coupling conductance. In -/- lenses the coupling conductance of MF was zero, and these fibers were depolarized by about 30 mV from normal (approximately -65 mV). The DF remained coupled, but the conductance was reduced to 30-35% of normal. However, the gap junctions in the DF of alpha3 -/- lenses remained sensitive to pH. We conclude that alpha3 connexin is necessary for the coupling of central fibers to peripheral cells, and that this coupling is essential for fiber cell homeostasis because uncoupled MF depolarize and subsequently become opaque.


A rapid and versatile method for the isolation, purification and cryogenic storage of Schwann cells from adult rodent nerves.
We herein developed a protocol for the rapid procurement of adult nerve-derived Schwann cells (SCs) that was optimized to implement an immediate enzymatic dissociation of fresh nerve tissue while maintaining high cell viability, improving yields and minimizing fibroblast and myelin contamination. This protocol introduces: (1) an efficient method for enzymatic cell release immediately after removal of the epineurium and extensive teasing of the nerve fibers; (2) an adaptable drop-plating method for selective cell attachment, removal of myelin debris, and expansion of the initial SC population in chemically defined medium; (3) a magnetic-activated cell sorting purification protocol for rapid and effective fibroblast elimination; and (4) an optional step of cryopreservation for the storage of the excess of cells. Highly proliferative SC cultures devoid of myelin and fibroblast growth were obtained within three days of nerve processing. Characterization of the initial, expanded, and cryopreserved cell products confirmed maintenance of SC identity, viability and growth rates throughout the process. Most importantly, SCs retained their sensitivity to mitogens and potential for differentiation even after cryopreservation. To conclude, this easy-to-implement and clinically relevant protocol allows for the preparation of expandable homogeneous SC cultures while minimizing time, manipulation of the cells, and exposure to culture variables.


Genetic enhancers of partial PLK1 inhibition reveal hypersensitivity to kinetochore perturbations.
Polo-like kinase 1 (PLK1) is a serine/threonine kinase required for mitosis and cytokinesis. As cancer cells are often hypersensitive to partial PLK1 inactivation, chemical inhibitors of PLK1 have been developed and tested in clinical trials. However, these small molecule inhibitors alone are not completely effective. PLK1 promotes numerous molecular and cellular events in the cell division cycle and it is unclear which of these events most crucially depend on PLK1 activity. We used a CRISPR-based genome-wide screening strategy to identify genes whose inactivation enhances cell proliferation defects upon partial chemical inhibition of PLK1. Genes identified encode proteins that are functionally linked to PLK1 in multiple ways, most notably factors that promote centromere and kinetochore function. Loss of the kinesin KIF18A or the outer kinetochore protein SKA1 in PLK1-compromised cells resulted in mitotic defects, activation of the spindle assembly checkpoint and nuclear reassembly defects. We also show that PLK1-dependent CENP-A loading at centromeres is extremely sensitive to partial PLK1 inhibition. Our results suggest that partial inhibition of PLK1 compromises the integrity and function of the centromere/kinetochore complex, rendering cells hypersensitive to different kinetochore perturbations. We propose that KIF18A is a promising target for combinatorial therapies with PLK1 inhibitors.


H2A.Z chaperones converge on histone H4 acetylation for melanoma cell proliferation.
High levels of H2A.Z promote melanoma cell proliferation and correlate with poor prognosis. However, the role of the two distinct H2A.Z histone chaperone complexes, SRCAP and P400-TIP60, in melanoma remains unclear. Here, we show that individual depletion of SRCAP , P400 , and VPS72 (YL1 ) not only results in loss of H2A.Z deposition into chromatin, but also a striking reduction of H4 acetylation in melanoma cells. This loss of H4 acetylation is found at the promoters of cell cycle genes directly bound by H2A.Z and its chaperones, suggesting a highly coordinated regulation between H2A.Z deposition and H4 acetylation to promote their expression. Knockdown of each of the three subunits downregulates E2F1 and its targets, resulting in a cell cycle arrest akin to H2A.Z depletion. However, unlike H2A.Z deficiency, loss of the shared H2A.Z chaperone subunit YL1 induces apoptosis. Furthermore, YL1 is overexpressed in melanoma tissues, and its upregulation is associated with poor patient outcome. Together, these findings provide a rationale for future targeting of H2A.Z chaperones as an epigenetic strategy for melanoma treatment.


Accessing Targeted Therapies: A Potential Roadblock to Implementing Precision Oncology?
Advances in genomic techniques have led to increased use of next-generation sequencing (NGS). We evaluated the extent to which these tests guide treatment decisions. We developed and distributed a survey assessing NGS use and outcomes to a survey pool of ASCO members. Comparisons between groups were performed with Wilcoxon two-sample, chi-square, and Fisher's exact tests. Among 178 respondents, 62% were male, 54% White, and 67% affiliated with academic centers. More than half (56%) indicated that NGS provided actionable information to a moderate or great extent. Use was highest (median ≥ 70% of cases) for lung and gastric cancer, and lowest (median < 25% of cases) in head and neck and genitourinary cancers. Approximately one third of respondents reported that, despite identification of an actionable molecular variant, patients were sometimes or often unable to access the relevant US Food and Drug Administration-approved therapy. When NGS did not provide actionable results, individuals reporting great or moderate guidance overall from NGS in treatment recommendations were more likely to request the compassionate use of an unapproved drug (P < .001), enroll on a clinical trial (P < .01), or treat off-label with a drug approved for another indication (P = .02). When NGS identifies an actionable result, a substantial proportion of clinicians reported encountering challenges obtaining approved therapies on the basis of these results. Perceived overall impact of NGS appears associated with clinical behavior unrelated to actionable NGS test results, including pursuing off-label or compassionate use of unapproved therapies or referring to a clinical trial.


Channeling and conformational changes in the heterotetrameric sarcosine oxidase from Corynebacterium sp. U-96.
We characterized the crystal structures of heterotetrameric sarcosine oxidase (SO) from Corynebacterium sp. U-96 complexed with methylthioacetate (MTA), pyrrole 2-carboxylate (PCA) and sulphite, and of sarcosine-reduced SO. SO comprises α-, β-, γ- and δ-subunits; FAD and FMN cofactors; and a large internal cavity. MTA and PCA are sandwiched between the re-face of the FAD isoalloxazine ring and the β-subunit C-terminal residues. Reduction of flavin cofactors shifts the β-subunit Ala1 towards the α-subunit Met55, forming a surface cavity at the oxygen-channel vestibule and rendering the β-subunit C-terminal residues mobile. We identified three channels connecting the cavity and the enzyme surface. Two of them exist in the inter-subunit space between α and β-subunits, and the substrate sarcosine seems to enter the active site through either of these channels and reaches the re-side of the FAD isoalloxazine ring by traversing the mobile β-subunit C-terminal residues. The third channel goes through the α-subunit and has a folinic acid-binding site, where the iminium intermediate is converted to Gly and either formaldehyde or, 5,10-methylenetetrahydrofolate. Oxygen molecules are probably located on the surface cavity and diffuse to the FMN isoalloxazine ring; the H(2)O(2) formed exits via the oxygen channel.


New insights into the by-product fatigue mechanism of the photo-induced ring-opening in diarylethenes.
The photochromic properties of diarylethenes, some of the most studied class of molecular switches, are known to be controlled by non-adiabatic decay at a conical intersection seam. Nevertheless, as their fatigue-reaction mechanism - leading to non-photochromic products - is yet to be understood, we investigate the photo-chemical formation of the so-called by-product isomer using three complementary computational methods (MMVB, CASSCF and CASPT2) on three model systems of increasing complexity. We show that for the ring-opening reaction a transition state on S1(2A) involving bond breaking of the penta-ring leads to a low energy S1(2A)/S0(1A) conical intersection seam, which lies above one of the transition states leading to the by-product isomer on the ground state. Therefore, radiationless decay and subsequent side-product formation can take place explaining the photo-degradation responsible for the by-product generation in diarylethene-type molecules. The effect of dynamic electron correlation and the possible role of inter-system crossing along the penta-ring opening coordinate are discussed as well.


Fibrinolytic activity of immobilized plasminogen activator.
Urokinase (UK) was immobilized onto the inner surface of 4-mm ID canine autogenous fibrocollagenous tubes in order to develop a fibrinolytic small-caliber vascular prosthesis. Using a glutaraldehyde entrapment process, the immobilized urokinase was compared to its soluble counterpart for its ability to degrade fibrin. Fibrin Degradation Product (FDP) levels increased with time for both soluble and immobilized urokinase, where FDP levels at complete lysis for both soluble and immobilized urokinase, where FDP levels at complete lysis were measured at 165.83 +/- 6.44 micrograms/ml. The rate of fibrinolysis was slower for the immobilized urokinase compared to its soluble form. The inhibitory effect of alpha-2 plasmin inhibitor was shown to be inimical to urokinase activity. Using a differential recirculation reactor apparatus and an esterolytic reaction involving N-alpha-acetyl-L-lysine methyl ester HCl, the amount of urokinase immobilized onto fibrocollagenous tubes (FCT) was measured to be 110 +/- 14 CTA units of UK per millimeter graft. The stability of immobilized urokinase was maintained for at least 75 hours of operation time, which corresponds to 1 year maintained for at least 75 hours of operation time, which corresponds to 1 year of storage time. Urokinase-bound fibrocollagenous tubes (UK-FCT), in addition to control FCT's, Perloff prostheses, and the autogenous saphenous vein, were interposed onto the carotid artery of canine mongrels using end-to-end anastomoses with 7-0 prolene sutures. Results indicate that the patency rates for UK-FCT's were 86% and 62% after 1 and 2 months, respectively. The Perloff graft and the control FCT had much lower patency rates. These results indicate that immobilizing urokinase is a viable method of producing a small-caliber biocompatible vascular prosthesis.


Electron transfer in neptunyl(VI)-neptunyl(V) complexes in solution.
The rates and mechanisms of the electron self-exchange between Np(V) and Np(VI) in solution have been studied with quantum chemical methods and compared with previous results for the U(V)-U(VI) pair. Both outer-sphere and inner-sphere mechanisms have been investigated, the former for the aqua ions, the latter for binuclear complexes containing hydroxide, fluoride, and carbonate as bridging ligand. Solvent effects were calculated using the Marcus equation for the outer-sphere reactions and using a nonequilibrium PCM method for the inner-sphere reactions. The nonequilibrium PCM appeared to overestimate the solvent effect for the outer-sphere reactions. The calculated rate constant for the self-exchange reaction NpO2(+)(aq) + NpO2(2+)(aq) right harpoon over left harpoon NpO2(2+)(aq) + NpO2(+)(aq), at 25 degrees C is k = 67 M(-1) s(-1), in fair agreement with the observed rates 0.0063-15 M(-1) s(-1). The differences between the Np(V)-Np(VI) and the U(V)-U(VI) pairs are minor.


"Filling the Gaps": Canadian Voluntary Nurses, the 1917 Halifax Explosion, and the Influenza Epidemic of 1918.
During the Great War, St. John Ambulance established a reserve of approximately 2,000 casually trained nursing volunteers as Voluntary Aid Detachment nurses, or VADs, who assisted in military hospitals at home and overseas. During the 1917 Halifax explosion, and the 1918 influenza epidemic, VADs also provided substantial assistance to qualified medical and nursing personnel. This paper considers their record during these specific events, arguing that despite the potential for VADs to undermine the status and job security of qualified graduate nurses, they proved instead both a valuable asset and a positive influence for the future development of Canadian nursing.


Smoothed particle hydrodynamic model for viscoelastic fluids with thermal fluctuations.
We present a fluid-particle model for a polymer solution in nonisothermal situations. The state of the fluid particles is characterized by the thermodynamic variables and a configuration tensor that describes the underlying molecular orientation of the polymer molecules. The specification of very simple physical mechanisms inspired by the dynamics of single polymer molecules allows one, with the help of the general equation for nonequilibrium reversible-irreversible coupling (GENERIC) formalism, to derive the equations of motion for a set of fluid particles carrying polymer molecules in suspension. In the simplest case of Hookean dumbbells we recover a fluid-particle version of the Oldroyd-B model in which thermal fluctuations are included consistently. Generalization to more complex viscoelastic models, such as finitely extensible nonlinear elastic Peterlin (FENE-P) model, with the proper introduction of thermal fluctuations is straightforward.


In situ analysis of the evolution of the primary immune response in murine Chlamydia trachomatis genital tract infection.
Adaptive immune responses contribute to the resolution of Chlamydia trachomatis genital tract infection and protect against reinfection, but our understanding of the mechanisms of those protective responses is incomplete. In this study, we analyzed by in situ immunohistochemistry the progression of the inflammatory and cytokine responses in the genital tracts of mice vaginally infected with C. trachomatis strain mouse pneumonitis. The cellular inflammatory response was characterized by an initial elevation in myeloid cells in the vagina (day 3) and uterine horns (day 7), followed by a marked rise in the number of T cells, predominantly CD4(+) cells. CD8(+) T cells and CD45R(+) B cells were also detected but were much less numerous. Perivascular clusters of CD4(+) T cells, which resembled clusters of T cells seen in delayed-type hypersensitivity responses, were evident by 2 weeks postinfection. Following the resolution of infection, few CD8(+) T cells and CD45R(+) B cells remained, whereas numerous CD4(+) T cells and perivascular clusters of CD4(+) T cells persisted in genital tract tissues. Interleukin-12 (IL-12)- and tumor necrosis factor alpha (TNF-alpha)-producing cells were observed in vaginal tissue by day 3 of infection and in uterine tissues by day 7. Cells producing IL-4 or IL-10 were absent from vaginal tissues at day 3 of infection but were present in uterine tissues by day 7 and were consistently more numerous than IL-12- and TNF-alpha-producing cells. Thus, the evolution of the local inflammatory response was characterized by the accumulation of CD4(+) T cells into perivascular clusters and the presence of cells secreting both Th1- and Th2-type cytokines. The persistence of CD4(+)-T-cell clusters long after infection had resolved (day 70) may provide for a readily mobilizable T-cell response by which previously infected animals can quickly respond to and control a secondary infectious challenge.


Observed strengthening of interbasin exchange via the Indonesian seas due to rainfall intensification.
A proxy of the Indonesian Throughflow (ITF) transport, developed using in situ hydrographic measurements along with assimilations, shows a significant strengthening trend during the past decade. This trend is due to a freshening and subsequent increase in the halosteric component of the ITF transport associated with enhanced rainfall over the Maritime Continent over the same period. The strengthening of the ITF transport leads to a significant change in heat and freshwater exchange between the Pacific and Indian Oceans and contributes to the warming and freshening of the eastern Indian Ocean. The combined effect of the ITF transport of mass and freshwater along with tropical rainfall plays a very important role in the climate system.


Purifying selection maintains highly conserved noncoding sequences in Drosophila.
The majority of metazoan genomes consist of nonprotein-coding regions, although the functional significance of most noncoding DNA sequences remains unknown. Highly conserved noncoding sequences (CNSs) have proven to be reliable indicators of functionally constrained sequences such as cis-regulatory elements and noncoding RNA genes. However, CNSs may arise from nonselective evolutionary processes such as genomic regions with extremely low mutation rates known as mutation "cold spots." Here we combine comparative genomic data from recently completed insect genome projects with population genetic data in Drosophila melanogaster to test predictions of the mutational cold spot model of CNS evolution in the genus Drosophila. We find that point mutations in intronic and intergenic CNSs exhibit a significant reduction in levels of divergence relative to levels of polymorphism, as well as a significant excess of rare derived alleles, compared with either the nonconserved spacer regions between CNSs or with 4-fold silent sites in coding regions. Controlling for the effects of purifying selection, we find no evidence of positive selection acting on Drosophila CNSs, although we do find evidence for the action of recurrent positive selection in the spacer regions between CNSs. We estimate that approximately 85% of sites in Drosophila CNSs are under constraint with selection coefficients (N(e)s) on the order of 10-100, and thus, the estimated strength and number of sites under purifying selection is greater for Drosophila CNSs relative to those in the human genome. These patterns of nonneutral molecular evolution are incompatible with the mutational cold spot hypothesis to explain the existence of CNSs in Drosophila and, coupled with similar findings in mammals, argue against the general likelihood that CNSs are generated by mutational cold spots in any metazoan genome.


Surveillance for hemodialysis access stenosis: usefulness of ultrasound vector volume flow.
To investigate if ultrasound vector-flow imaging (VFI) is equal to the reference method ultrasound dilution technique (UDT) in estimating volume flow and changes over time in arteriovenous fistulas (AVFs) for hemodialysis. From January 2014 to January 2015, patients with end-stage renal disease and matured functional AVFs were consecutively solicited to participate in this prospective study. All patients were included after written informed consent and approval by the National Committee on Biomedical Research Ethics and the local Ethics Committee (journal no. H-4-2014-FSP). VFI and UDT measurements were performed monthly over a six-month period. Nineteen patients were included in the study. VFI measurements were performed before dialysis, and UDT measurements after. Statistical analyses were performed with Bland-Altman plot, Student's t-test, four-quadrant plot, and regression analysis. Repeated measurements and precision analysis were used for reproducibility determination. Precision measurements for UDT and VFI were 32% and 20%, respectively (p = 0.33). Average volume flow measured with UDT and VFI were 1161 mL/min (±778 mL/min) and 1213 mL/min (±980 mL/(min), respectively (p = 0.3). The mean difference was -51 mL/min (CI: -150 mL/min to 46 mL/min) with limits of agreement from -35% to 54%, with a strong correlation (r2 = 0.87). A large change in volume flow between dialysis sessions detected by UDT was confirmed by VFI (p = 0.0001), but the concordance rate was poor (0.72). VFI is an acceptable method for volume flow estimation and volume flow changes over time in AVFs.


Dengue.
Dengue is a vector-borne viral infection that endangers an estimated 2.5 billion people. Disease caused by dengue ranges from a relatively minor febrile illness to a life-threatening condition characterized by extensive capillary leak. A greater understanding of dengue has the potential to improve both the clinical management of individual cases and the control of the disease. We searched the available literature using PubMed, Embase and Web of Science for relevant articles and abstracts. Addressing our gaps in the understanding of disease pathogenesis and improving our knowledge of dengue virus biology are necessary in order to develop tools to effectively control, diagnose and treat the disease. The pathogenesis of dengue is multifactorial and depends on both host and virus factors. A more integrated understanding of disease pathogenesis is necessary. There are many questions related to disease pathogenesis, development of diagnostics, drug and vaccine development and individual case management that need addressing if the disease is to be successfully tackled.


The use of analytical isoelectric focusing for detection and identification of beta-lactamases.
BETA-Lactamases (EC. 188.8.131.52) from strains of Gram-negative bacteria have been studied using analytical isoelectric focusing. This permits a visual comparison of the patterns of beta-lactamase bands produced by enzymes from different organisms. Purification of crude intracellular preparations is unnecessary and the technique is sufficiently sensitive to demonstrate beta-lactamase in mutants previously reported to lack the enzyme. R that have not been distinguished from one another biochemically or immunologically can be differentiated by isoelectric focusing. Conversely, the enzymes specified by the R factors RTEM, R1 and RGN14, with identical isoelectric focusing patterns have the same biochemical properties. Chromosomal and R-factor-mediated beta-lactamases from single strains have been separated and their identities confirmed by immunoisoelectric focusing. R factor-mediated enzymes gave identical isoelectric focusing patterns irrespective of the host strain. Isoelectric focusing can therefore be used to observe the transfer of beta-lactamases carried by R factors.


Loss of a homologous group of proteins in a dominantly inherited ectodermal malformation.
Hair from mice bearing the dominantly inherited Naked trait (NN) and from normal (NN) mice of the same inbred strain was separated into its major protein components by standard techniques. The relative amounts of proteins in these components were then determined by a regression method from the amino acid composition of the hair samples and of the fractions into which they had been separated. The results indicated that the amount of soluble fibril in Naked-mouse hair is decreased. Polyacrylamide-gel electrophoresis of this fraction prepared from the hair of both normal and Naked mice revealed that all protein bands present in the normal are also present in the Naked mice. However, a densitometric scan of the gels at 280 nm showed that the soluble fibril fraction from Naked-mouse hair is deficient in several proteins which, on amino acid analysis, were found to contain 31% glycine and 10% tyrosine. Gel filtration of S-carboxymethylkerateine prepared from normal and mutant hair showed that the mutant hair is deficient in a heterogeneous, low-molecular-weight fraction also rich in glycine and tyrosine. Our present data do not reveal the mechanism whereby a single gene locus modulates the production of several different proteins.


Severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice.
The spike protein (S), a membrane component of severe acute respiratory syndrome coronavirus (SARS-CoV) is anticipated to be an important component of candidate vaccines. We constructed recombinant forms of the highly attenuated modified vaccinia virus Ankara (MVA) containing the gene encoding full-length SARS-CoV S with and without a C-terminal epitope tag called MVA/S-HA and MVA/S, respectively. Cells infected with MVA/Sor MVA/S-HA synthesized a 200-kDa protein, which was recognized by antibody raised against a synthetic peptide of SARS-CoV S or the epitope tag in Western blot analyses. Further studies indicated that S was N-glycosylated and migrated in SDS polyacrylamide gels with an apparent mass of approximately 160 kDa after treatment with peptide N-glycosidase F. The acquisition of resistance to endoglycosidase H indicated trafficking of S to the medial Golgi compartment, and confocal microscopy showed that S was transported to the cell surface. Intranasal or intramuscular inoculations of BALB/c mice with MVA/S produced serum antibodies that recognized the SARS S in ELISA and neutralized SARS-CoV in vitro. Moreover, MVA/S administered by either route elicited protective immunity, as shown by reduced titers of SARS-CoV in the upper and lower respiratory tracts of mice after challenge. Passive transfer of serum from mice immunized with MVA/S to naïve mice also reduced the replication of SARS-CoV in the respiratory tract after challenge, demonstrating a role for antibody to S in protection. The attenuated nature of MVA and the ability of MVA/S to induce neutralizing antibody that protects mice support further development of this candidate vaccine.


Carriage of Extended-Spectrum Beta-Lactamase-Plasmids Does Not Reduce Fitness but Enhances Virulence in Some Strains of Pandemic E. coli Lineages.
Pathogenic ESBL-producing E. coli lineages occur frequently worldwide, not only in a human health context but in animals and the environment, also in settings with low antimicrobial pressures. This study investigated the fitness costs of ESBL-plasmids and their influence on chromosomally encoded features associated with virulence, such as those involved in the planktonic and sessile behaviors of ST131 and ST648 E. coli. ESBL-plasmid-carrying wild-type E. coli strains, their corresponding ESBL-plasmid-"cured" variants (PCV), and complementary ESBL-carrying transformants were comparatively analyzed using growth curves, Omnilog® phenotype microarray (PM) assays, macrocolony and biofilm formation, swimming motility, and RNA sequence analysis. Growth curves and PM results pointed toward similar growth and metabolic behaviors among the strains. Phenotypic differences in some strains were detected, including enhanced curli fimbriae and/or cellulose production as well as a reduced swimming capacity of some ESBL-carrying strains, as compared to their respective PCVs. RNA sequencing mostly confirmed the phenotypic results, suggesting that the chromosomally encoded csgD pathway is a key factor involved. These results contradict the hypothesis that ESBL-plasmid-carriage leads to a fitness loss in ESBL-carrying strains. Instead, the results indicate an influence of some ESBL-plasmids on chromosomally encoded features associated with virulence in some E. coli strains. In conclusion, apart from antibiotic resistance selective advantages, ESBL-plasmid-carriage may also lead to enhanced virulence or adaption to specific habitats in some strains of pandemic ESBL-producing E. coli lineages.


Hypertension in the Kidney Transplant Recipient: Overview of Pathogenesis, Clinical Assessment, and Treatment.
Cardiovascular disease is the leading cause of death in patients with chronic renal disease and the most common cause of death and allograft loss among kidney transplant recipients. Transplant patients often have multiple cardiovascular risk factors antedating transplantation. Among the most prominent is hypertension (HTN), which affects at least 90% of transplant patients. Uncontrolled HTN is an independent risk factor for allograft loss. The etiology of HTN in transplant recipients is complex and multifactorial, including the use of essential immunosuppressive medications. Post-transplant HTN management requires a systematic and individualized approach with nonpharmacologic and pharmacologic therapies. There is no single ideal agent or treatment algorithm. Patients should regularly monitor and record their blood pressure at home. Often, multiple antihypertensive drugs are needed to achieve a goal blood pressure of 120-140/70-90 mm Hg. As transplant recipients commonly must take 8 to 12 different medications daily, adherence must be continually encouraged and monitored. Special attention must be paid to potential drug side effects and drug interactions with immunosuppressive medications.


Ethanol inhibits insulin action on lipolysis and on insulin release in elderly men.
We have studied effects of ethanol on insulin's ability to suppress its own release and on its antilipolytic action in 12 healthy elderly men during euglycemic hyperinsulinemia. Insulin secretion was estimated from plasma C-peptide concentrations. Lipolysis was determined with the two stable isotopes [2H5]glycerol and [1-13C]palmitate. Hyperinsulinemia (approximately 350 pM) decreased plasma C-peptide by approximately 60% (from 325 to 122 pM, P < 0.05). Ethanol (approximately 10 mM) completely prevented the fall in C-peptide concentration. Ethanol decreased the antilipolytic action of insulin by approximately 40% [with insulin alone, glycerol rate of appearance (Ra) decreased from 1.8 to 0.6 mumol.kg-1 x min-1; with insulin + ethanol, it only decreased from 1.8 to 1.1 mumol.kg-1 x min-1]. Ethanol did not affect palmitate Ra, which fell from 1.4 to 0.6 mumol.kg-1 x min-1 with insulin and from 1.4 to 0.3 mumol.kg-1 x min-1 with insulin plus ethanol. Fatty acid reesterification was not affected by insulin but tripled (from 0.6 to 1.9 mumol.kg-1 x min-1) in response to insulin plus ethanol. Our data showed that modest concentrations of ethanol suppressed the inhibitory actions of insulin on its own release and on lipolysis. The inhibition by ethanol of various insulin actions, including glucose disposal, lipolysis, and insulin release, in diverse tissues such as muscle, adipose tissue, and pancreas raises the possibility that ethanol may produce a state of generalized insulin resistance.


Designing an internal organizational merger.
Healthcare managers face problems resulting from restructuring and downsizing activities meant to increase operational efficiency and reduce costs. Although keys to the success of multiorganizational mergers are well documented, directives for managing internal mergers are less clearly stated. The authors discuss a five-step plan designed to facilitate an internal merger through the strategic coordination of stakeholders.


Tunneling inhibition for subwavelength light.
We show that light tunneling inhibition may take place in suitable dynamically modulated waveguide arrays for light spots whose features are remarkably smaller than the wavelength of light. We found that tunneling between neighboring waveguides can be suppressed for specific frequencies of the out-of-phase refractive index modulation, affording undistorted propagation of the input subwavelength light spots over hundreds of Rayleigh lengths. Tunneling inhibition turns out to be effective only when the waveguide separation in the array is above a critical threshold. Inclusion of a weak focusing nonlinearity is shown to improve localization. We analyze the phenomenon in purely dielectric structures and also in arrays containing periodically spaced metallic layers.


Quantitative analysis of periodontal defects in a skull model by subtraction radiography using a digital imaging device.
This paper describes a quantitative study of periodontal defects produced in a skull model using subtraction images obtained with a digital imaging device, radiovisiography (RVG). Analysis using radiovisiography was compared to changes in weight and volume determined by physical measurements. Four types of periodontal defects were studied: 2-wall, 3-wall, crater, and furcation. All defects were fabricated on posterior teeth in alveolar bone and done with triplicate samples. Defects were made progressively larger in approximately 1 mm steps allowing examination of defects ranging from 1 mm to 5 mm. Radiographic images before and after each step were obtained with a radiovisiography system and subtracted. Changes in weight and defect volume were also recorded and compared to the results obtained after each step from the digital subtraction. For each defect type, a regression analysis was performed to compare changes in bone mass determined by weight with calculated bone loss determined by subtraction radiography or by volume measurements. When the calculated bone losses were compared to the true bone losses it was evident that the subtraction method frequently, but not always, underestimated the lesion sizes. For all lesions the average underestimation was 22%. The largest underestimation occurred with furcation lesions where the measured bone loss was underestimated on average by 67%. Two-walled lesions were underestimated by 30%, 3-wall lesions by 3%, and crater lesions were overestimated by 10%. Furthermore, the accuracy of each 1 mm step in bone loss varied considerably. At present, the imaging system is not sufficiently accurate to establish absolute determinations of the bone loss, but would be clinically useful in determining relative changes in bone loss or gain after treatment. In addition, caution must be taken in interpreting a given change in calculated bone loss, since considerable variation may result in either underestimation or overestimation of bone loss.


Prenatal diagnosis of partial trisomy 21 associated with maternal balanced translocation 46xx der 21 t(21q;22q) with pericentric inversion of chromosome 9.
This communication reports prenatal diagnosis of partial trisomy 21 resulting from balanced translocation (21q;22q) in a 36-year-old gravida 7, para 1 woman. The lady had only one living child and there was history of recurrent spontaneous first trimester abortions. Triple test was abnormal in the present conception. In addition, the woman had pericentric inversion of chromosome 9, a finding scarcely reported previously with carrier status in Indian literature. A few cytogeneticists consider this as a normal variant. However, many reports in the recent literature link pericentric inversion of chromosome 9 with infertility, recurrent abortions and a number of other abnormal conditions. A review of the relevant literature pertinent to the case is provided.


Expression and localization of insulin receptor in rat dorsal root ganglion and spinal cord.
The expression and localization of the insulin receptor (IR) was examined in rat dorsal root ganglia (DRG) and spinal cord using Western blotting, in situ hybridization and immunocytochemistry. Western blotting showed that the molecular weight of the IR beta subunit was higher in PNS than that found in CNS. Both IR mRNA and protein expressions were highest in small-sized sensory DRG neurons and myelinated sensory root fibers expressed higher levels of IR protein than myelinated anterior root fibers. In the spinal cord, IR immunoreactive neurons were present in lateral lamina V and in lamina X, suggesting the presence of IR in nociceptive pathways. Electronmicroscopy of DRGs revealed a polarized localization of the IR in abaxonal Schwann cell membranes, outer mesaxons in close vicinity to tight junctions of both myelinating and non-myelinating Schwann cells and to plasma membranes of sensory neurons. From these findings, we speculate that insulin may play a role in sensory fibers involved in nociceptive function often perturbed in diabetic neuropathy. The high expression of IR localizing to tight junctions of dorsal root mesaxons of DRGs may suggest a regulatory role on barrier functions compensating for the lack of a blood-nerve barrier in dorsal root ganglia. This is consistent with the colocalization of IR with tight junctions of the paranodal barrier and endoneurial endothelial cells in peripheral nerve.


MicroRNA-367-3p directly targets RAB23 and inhibits proliferation, migration and invasion of bladder cancer cells and increases cisplatin sensitivity.
This study investigated the biological role of miR-367-3p upregulation in bladder cancer and verified the mutual relation between miR-367-3p and RAB23. Expression levels of miR-367-3p were determined by RT-qPCR in bladder cancer cell lines and human bladder cancer tissues. The effects of miR-367-3p on proliferation, migration and invasion were evaluated by cell colony formation assays, wound healing assays and trans-well assays, respectively. The effects of miR-367-3p and RAB23 on cisplatin sensitivity of bladder cancer cells were assessed by CCK-8 assay. The expression of its target-RAB23 was determined by western blotting in T24, 5637. Plasmids used in dual-luciferase assays were constructed to confirm the action of miR-367-3p on downstream target-RAB23 in T24 cells. And also, the role of miR-367-3p in tumorigenesis was also confirmed in nude mouse models. The downregulation of miR-367-3p was observed in human bladder cancer tissues. MiR-367-3p downregulation positively correlated with tumor stage and tumor grade. MiR-367-3p overexpression in T24, 5637 cells suppressed the proliferation, migration, and invasion of bladder cancer cells in vitro while decreasing IC50 values under T24 and 5637 cisplatin treatment conditions. RAB23 was shown to be upregulated in bladder cancer tissues and cell lines. MiR-367-3p directly bound to the 3' UTR of RAB23 in T24 cells. RAB23 was potentially accounted for the aforementioned functions of miR-367-3p. Tumor formation experiments in nude mouse models confirmed that overexpression of miR-367-3p could inhibit tumor growth and invasion in vivo. miR-367-3p acts as a tumor suppressor in bladder cancer by downregulating RAB23 signaling. We conjecture that miR-367-3p-mediated downregulation of RAB23 expression may be a new therapeutic strategy for bladder cancer treatment.


The hemodynamics of isoproterenol-induced cardiac failure in the rat.
In virgin, male Sprague-Dawley rats, subcutaneous injections of 2.5 mg/kg or 250 mg/kg isoproterenol increased heart rate and aortic dF/dt and decreased total peripheral resistance. The net systemic response was an arterial hypotension. The larger dose of isoproterenol initially produced a greater hypotension; reflex compensatory responses followed. Cardiac failure occurred by 24 hours post-isoproterenol. The extent of cardiac failure was isoproterenol dose dependent. By gross inspection of the epicardial surface of the hearts of the isoproterenol-treated rats, anatomical injury also appeared to be isoproterenol dose dependent. The data presented in this study support the existing theory that isoproterenol-induced myocardial damage is due to a relative myocardial hypoxia produced by artereial hypotension and myocardial hyperactivity. The data also indicate that reflex responses to arterial hypotension occur and may be detrimental. Cardiac failure is produced by massive quantities of isoproterenol, and the degree of cardiac failure is dose dependent.


The Value Of The Nonprofit Hospital Tax Exemption Was $24.6 Billion In 2011.
The federal government encourages public support for charitable activities by allowing people to deduct donations to tax-exempt organizations on their income tax returns. Tax-exempt hospitals are major beneficiaries of this policy because it encourages donations to the hospitals while shielding them from federal and state tax liability. In exchange, these hospitals must engage in community benefit activities, such as providing care to indigent patients and participating in Medicaid. The congressional Joint Committee on Taxation estimated the value of the nonprofit hospital tax exemption at $12.6 billion in 2002--a number that included forgone taxes, public contributions, and the value of tax-exempt bond financing. In this article we estimate that the size of the exemption reached $24.6 billion in 2011. The Affordable Care Act (ACA) brings a new focus on community benefit activities by requiring tax-exempt hospitals to engage in communitywide planning efforts to improve community health. The magnitude of the tax exemption, coupled with ACA reforms, underscores the public's interest not only in community benefit spending generally but also in the extent to which nonprofit hospitals allocate funds for community benefit expenditures that improve the overall health of their communities.


Association between lower lobe location and upstaging for early-stage non-small cell lung cancer.
To identify factors associated with a misclassification of the true disease stage by comparing the differences between the clinical and pathologic stage of patients with early-stage non-small cell lung cancer (NSCLC). A prospective cohort study. A multidisciplinary thoracic oncology clinic at a university-affiliated Veterans Affairs medical center. One hundred nine male veterans with clinical stage I/II NSCLC who had undergone thoracotomy with systematic lymph node dissection. Prospective data were collected on all patients between September 1997 and April 2002. Logistic regression analysis was used to establish the odds ratio (OR) for predictors of changes in stage. A stage misclassification was found in 35.8% of patients (39 of 109 patients) after thoracotomy with lymph node dissection, and all but one patient were upstaged. Unsuspected nodal involvement (N stage) resulted in the upstaging of 16.5% of the patients, a change in tumor stage (T stage) resulted in the upstaging of 13.8% of the patients, a change in both stages resulted in the upstaging of 2.7% of patients, and the designation of metastatic disease resulted in the upstaging of 1.9% of the patients. The rate of unsuspected mediastinal lymph node involvement (pathologic stage N2) was 8.3% (9 of 109 patients), despite negative mediastinoscopy findings. Complete anatomic resection was performed in all patients. Advanced disease was found in 8.3% of the patients (9 of 109 patients) [stage IIIB or IV]. Having the primary tumor in a lower lobe location was the only statistically significant factor associated with upstaging (OR, 3.56; 95% confidence interval, 1.4 to 9.1). The effect of location was robust after controlling for tumor size and the prior performance of mediastinoscopy. Patient age, smoking history, weight loss, tumor size, and tumor histology were all found not to be associated with upstaging. A lower lobe tumor location in patients with early-stage NSCLC appears to be associated with upstaging after surgery. We conclude that a tumor location in a lower lobe deserves special attention.


Effect of Tomato Nutrient Complex on Blood Pressure: A Double Blind, Randomized Dose⁻Response Study.
Oxidative stress is implicated in the pathogenesis of essential hypertension, a risk factor for cardiovascular morbidity and mortality. Tomato carotenoids such as lycopene and the colorless carotenoids phytoene and phytofluene induce the antioxidant defense mechanism. This double-blind, randomized, placebo-controlled study aimed to find effective doses of Tomato Nutrient Complex (TNC) to maintain normal blood pressure in untreated hypertensive individuals. The effect of TNC treatment (5, 15 and 30 mg lycopene) was compared with 15 mg of synthetic lycopene and a placebo over eight weeks. Results indicate that only TNC treatment standardized for 15 or 30 mg of lycopene was associated with significant reductions in mean systolic blood pressure (SBP). Treatment with the lower dose standardized for 5 mg of lycopene or treatment with 15 mg of synthetic lycopene as a standalone had no significant effect. To test carotenoid bioavailability, volunteers were treated for four weeks with TNC providing 2, 5 or 15 mg lycopene. The increase in blood levels of lycopene, phytoene, and phytofluene was dose dependent. Results suggest that only carotenoid levels achieved by the TNC dose of 15 mg lycopene or higher correlate to a beneficial effect on SBP in hypertensive subjects while lower doses and lycopene alone do not.


Antigen receptor signal transduction: activating and inhibitory antigen receptors regulate STAT1 serine phosphorylation.
Antigen receptors are crucial regulators of the mammalian immune response. Immediate antigen receptor proximal signal transduction pathways mediated by tyrosine (Tyr) kinases are well defined. In contrast, much less is known about the network of serine (Ser) kinases and Ser kinase substrates that are linked to antigen receptor function. Here we describe a new signaling module for antigen receptors in lymphocytes; a Ser kinase pathway that phosphorylates Ser 727 in STAT1alpha, a member of the signal transducer and activator of transcription gene family. In the present study we have explored the regulation of STAT1 Ser 727 phosphorylation in human T and B lymphocytes and show that it is controlled by both positive and negative antigen receptor signaling cascades. Ligation of antigen receptors in both B and T cells induce a delayed but then sustained phosphorylation of STAT1 on Ser 727. STAT1 Ser phosphorylation is induced by the TCR in the absence of STAT1 Tyr phosphorylation, indicating that in T cells STAT1 Ser and Tyr phosphorylation are independent events. Antigen receptor regulation of STAT Ser phosphorylation is dependent on phosphatidylinositol 3-kinase-mediated signals. Furthermore, the negative regulatory receptor FcgammaRIIb, which mediates vital feedback control of B cell responses, prevents antigen receptor-induced phosphorylation of STAT1 Ser 727. The ability of antigen receptors to both positively and negatively regulate STAT1 Ser 727 phosphorylation reveals a Ser kinase network that operated during sustained responses to antigen receptor engagement.


[Plasmacytoma of the thyroid. A review apropos a case].
Isolated presentation of outer bone marrow plasmatic cell neoplasia are non-frequent (less than 10% of all plasmacytomas). Thyroid gland involvement is rarely described. A case where immunohistochemistry confirmed the monoclonal type of proliferative cell is presented. Clinical, pathological and diagnosis criteria, as well as response to treatment, are reviewed.


Impact of urbanization on soil loss: a case study from sod production.
The rapidly increasing population of urban centers leads to the increasing need for greenspaces. Sodding of turfgrass provides instant greenspace, but it removes soil from sod farms. The extent of such removal has not been widely quantified. The amount quantity of soil and organic matter lost with sod harvest and the associated cost of nutrients lost from six sod farms in the Marmara region of Turkey were determined. Soil loss ranged from 166 to 243 Mg ha-1 year-1, while the associated organic matter loss ranged from 1 to 6 Mg ha-1 year-1. The amount of soil loss increased with increases in gravimetric water, clay, and silt contents, and duration under sod harvest, while it decreased with an increase in sand content. Annual nutrient lost ranged from 117 to 449 kg ha-1 for N, from 2 to 18 kg ha-1 for P2O5, and from 21 to 175 kg ha-1 for K2O. Replacing the nutrient lost would cost about $134 ha-1 year-1 for sandy soils and $444 ha-1 year-1 for fine-textured soils. Soil lost with sod harvest was 134 times higher than that from agricultural lands by erosion in the region, although the area under sod production is much smaller than that under croplands. Similarly, organic matter loss was 4 to 5 times higher than the accumulation rate under established turfgrass in golf courses and lawns in locations with similar climate. Overall, sod harvesting results in significant and costly soil, organic matter, and nutrient loss, which, although small in area, can be an important component of total soil erosion.


[Human low-density lipoproteins are peroxidized by free radicals via chain reactions triggered by the superoxide radical].
Low-density lipoproteins (LDL) oxidized by oxygen radicals (OR) are a potent atherogenic stimulus. Chemically modified LDL are internalized by macrophages via a specific cell surface receptor that was termed the scavenger receptor, and may induce foam cells transformation. A free radical is any chemical species that has an unpaired electron. This property renders it highly chemically reactive. When a radical reacts with a non radical another free radical is generated. This characteristic enables radicals to trigger chain reactions. Oxygen radicals are: superoxide anion (.O2-), hydroxyl radical (.OH) and hydrogen peroxide (H2O2). It is unknown whether LDL are modified via direct lipid oxidation by OR, or whether LDL are subsequently oxidized via chain reactions after initial OR attack. To distinguish between these 2 mechanisms, LDL were exposed to OR formed by xanthine/xanthine oxidase (X/XO). Peroxidation was measured from malonyldialdehyde (MDA) levels. Parallel experiments were performed in presence of the superoxide radical scavenger superoxide dismutase (SOD; 330 U/ml), or the hydrogen peroxide scavenger catalase (CAT; 1000 U/ml), or by adding the chain-reaction inhibitor butylhydroxytoluene (BHT; 1 mM) at selected time points. SOD, but not CAT prevented LDL peroxidation, indicating an obligatory role for superoxide radicals. Superoxide generation in this model lasts only a few minutes, however, MDA levels continued to increase over several hours. Furthermore, this phenomenon was blocked when BHT was added at various times after X/XO. These data show that LDL peroxidation is triggered by initial OR generation but then involves chain reactions which do not require continuous exposure to OR.(ABSTRACT TRUNCATED AT 250 WORDS)


Improved Thiéry staining for the ultrastructural detection of polysaccharides.
The use of silver vitellinate instead of silver proteinate in the Thiéry's staining for the ultrastructural detection of polysaccharides has advantages which derive from the lesser reactivity and higher stability of silver vitellinate solutions, allowing the obtention of a finer staining of periodic acid-Schiff (PAS)-positive materials with a cleaner background. The proposed method is particularly useful for the staining of PAS-positive cellular components at high resolution.


Valve failure following homograft aortic valve replacement: does implantation technique have an effect?
Structural valve deterioration (SVD) limits the long-term durability of homograft aortic valve replacement (AVR). Valves are implanted predominantly using two techniques, the free-hand sub-coronary (SC) technique or aortic root replacement (RR). Our objective was to identify risk factors associated with the development of SVD or ascending aortic dilatation. In particular we strived to determine whether the mode of implantation had an independent effect. Demographic and pre-operative clinical data were obtained retrospectively through case-note review. All operations were performed by a single surgeon. Actuarial freedom from >or=2+ AR (aortic regurgitation), elevated trans-valvular gradient (TVG) (>or=25 mmHg) and ascending aortic dilatation (>or=4.0 cm) were assessed using Kaplan-Meier curves and multivariable Cox proportional hazards regression. A propensity analysis was carried out using a non-parsimonius logistic regression model for implantation with SC vs. RR. Between 1 January 1991 and 1 January 2001, 215 patients underwent AVR with a homograft. The SC technique was used in 131 (61%) patients and 84 (39%) patients underwent RR. Technique was not an independent predictor for >or=2+ AR (adjusted hazard ratio 1.9; 95% CI 0.56-6.16, P = 0.31), elevated TVG (adjusted hazard ratio; 0.99; 95% CI 0.15-6.71, P = 0.99) or ascending aortic dilatation (adjusted hazard ratio 2.01; 95% CI 0.50-8.25, P = 0.33). One and 5 year actuarial freedom from >or=2+ AR (log-rank - P = 0.09) and ascending aortic dilatation (log-rank - P = 0.88) were not significantly different between groups. The incidence of SVD and ascending aortic dilatation is not affected by the method of implantation of the aortic homograft. All homografts are prone to SVD which is responsible for a progressive increase in the prevalence of these changes over time.


Marked peripheral eosinophilia due to prolonged administration of posaconazole.
Introduction. Posaconazole is a triazole antifungal that is used in the treatment of a variety of fungal infections, as well as in the management of mucormycosis (on an off-label basis). Eosinophilia associated with exposure to azole antifungals has been described rarely in the literature. Case presentation. A 31-year-old male on peritoneal dialysis (PD) for end-stage renal disease, secondary to diabetic nephropathy, presented to hospital with abdominal pain after a trip to St Lucia. He was taken to the operating room, where the PD catheter was removed and an abdominal-wall abscess was debrided. Rhizopus species was recovered on culture of the abdominal-wall tissue, and the patient was started on amphotericin B deoxycholate. He was subsequently stepped down to posaconazole, for a planned treatment duration of 12 months. Approximately 43 days after the initiation of posaconazole, it was noted that his peripheral eosinophil count started to rise. No other cause for the eosinophilia was identified. Posaconazole was discontinued, and the patient's eosinophil count began to drop 2 days later. The temporal association of eosinophilia following initiation of posaconazole and the subsequent improvement after drug discontinuation suggests a probable causal relationship. Conclusion. At the time of writing, there have been only two other published cases of azole-associated peripheral eosinophilia. In reporting this case, we hope to increase health-care provider awareness of this rare adverse event. For patients receiving prolonged therapy with posaconazole, periodic monitoring of the complete blood count with differential may be considered.


Eradication of Helicobacter pylori in patients with duodenal ulcer lowers basal and peak acid outputs to gastrin releasing peptide and pentagastrin.
Patients with duodenal ulcer (DU) have high basal (BAO) and peak (PAO) acid outputs. The effect of Helicobacter pylori eradication on these variables is unclear. To discover if gastric acid hypersecretion in patients with DU is caused by H pylori. BAO, gastrin releasing peptide (GRP), and pentagastrin stimulated PAO in 10 H pylori negative controls, and in 10 H pylori positive patients with DU was measured before and six months after H pylori eradication. H pylori status was determined by histology, culture, and by the 13C-urea breath test. After collecting a 30 minute basal aspirate, GRP 40 pmol/kg/h was infused for 45 minutes, and after a 30 minute washout, pentagastrin 6 micrograms/kg was injected intramuscularly. Basal and stimulated acid output (PAOGRP and PAOPg) were significantly higher in H pylori positive DU than in H pylori negative controls. Six months after H pylori eradication, basal and stimulated acid outputs were all significantly lower than before H pylori eradication. This study has shown that BAO, PAOGRP, and PAOPg are higher in H pylori positive DU than in H pylori negative controls. All decreased significantly six months after H pylori eradication, to fall within the range of controls. These results are compatible with a hypothesis that acid hypersecretion in duodenal ulcer disease is caused by H pylori infection.


Placental CRH as a Signal of Pregnancy Adversity and Impact on Fetal Neurodevelopment.
Early life is a period of considerable plasticity and vulnerability and insults during that period can disrupt the homeostatic equilibrium of the developing organism, resulting in adverse developmental programming and enhanced susceptibility to disease. Fetal exposure to prenatal stress can impede optimum brain development and deranged mother's hypothalamic-pituitary-adrenal axis (HPA axis) stress responses can alter the neurodevelopmental trajectories of the offspring. Corticotropin-releasing hormone (CRH) and glucocorticoids, regulate fetal neurogenesis and while CRH exerts neuroprotective actions, increased levels of stress hormones have been associated with fetal brain structural alterations such as reduced cortical volume, impoverishment of neuronal density in the limbic brain areas and alterations in neuronal circuitry, synaptic plasticity, neurotransmission and G-protein coupled receptor (GPCR) signalling. Emerging evidence highlight the role of epigenetic changes in fetal brain programming, as stress-induced methylation of genes encoding molecules that are implicated in HPA axis and major neurodevelopmental processes. These serve as molecular memories and have been associated with long term modifications of the offspring's stress regulatory system and increased susceptibility to psychosomatic disorders later in life. This review summarises our current understanding on the roles of CRH and other mediators of stress responses on fetal neurodevelopment.


Differential expression of specific FGF ligands and receptor isoforms during osteogenic differentiation of mouse Adipose-derived Stem Cells (mASCs) recapitulates the in vivo osteogenic pattern.
The ability of Adipose-derived Stem Cells (ASCs) to differentiate into various tissues in vitro and in vivo, a function known as "stem cell plasticity", makes them an appealing cell source for tissue engineering. Our laboratory is particularly focused on the potential role of adipose tissue as a readily available postnatal source of osteoprogenitor. Fibroblast growth factors (FGF) and their receptors (FGFR) are important regulators of osteogenesis. The goal of this study was to elucidate how changes in temporal expression patterns of individual components of the fibroblast growth factor (FGF) signaling axis correlate with osteogenic differentiation of mASCs. Our results indicate that FGF ligand genes, such as Fgf-2, -4, -8, and -18, displayed a differential and dynamic profile during mouse ASC (mASC) osteogenesis. Fgf-2 transcript was down-regulated, while Fgf-18 transcript level was strongly up-regulated. Interestingly, a drift in the ratio of different FGF-2 protein forms, with translation favoring the HMWFGF-2 forms, occurred during osteogenic differentiation, whereas, the expression of LMWFGF-2 form was down-regulated. This finding shares similarity with a previous study suggesting that preferential expression of the HMWFGF-2 forms is associated with a more osteogenic differentiated state of calvarial osteoblast. Moreover, a differential expression of Fgf Receptor 1 and 2 resembling that previously found in in vivo osteogenic study was observed. Thus, mASCs undergoing osteogenesis recapitulate the in vivo osteogenic differentiation expression pattern of FGF ligands and receptors of calvarial mesenchymal cells during their own osteogenic differentiation. Indeed, this observation further validates ASCs as a suitable resource for skeletal tissue engineering.


IL-9-triggered lncRNA Gm13568 regulates Notch1 in astrocytes through interaction with CBP/P300: contribute to the pathogenesis of experimental autoimmune encephalomyelitis.
Interleukin 9 (IL-9), produced mainly by T helper 9 (Th9) cells, has been recognized as an important regulator in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Astrocytes respond to IL-9 and reactive astrocytes always associate with blood-brain barrier damage, immune cell infiltration, and spinal injury in MS and EAE. Several long non-coding RNAs (lncRNAs) with aberrant expression have been identified in the pathogenesis of MS. Here, we examined the effects of lncRNA Gm13568 (a co-upregulated lncRNA both in EAE mice and in mouse primary astrocytes activated by IL-9) on the activation of astrocytes and the process of EAE. In vitro, shRNA-recombinant lentivirus with glial fibrillary acidic protein (GFAP) promoter were performed to determine the relative gene expression and proinflammatory cytokines production in IL-9 treated-astrocytes using Western blot, real-time PCR, and Cytometric Bead Array, respectively. RIP and ChIP assays were analyzed for the mechanism of lncRNA Gm13568 regulating gene expression. Immunofluorescence assays was performed to measure the protein expression in astrocytes. In vivo, H&E staining and LFB staining were applied to detect the inflammatory cells infiltrations and the medullary sheath damage in spinal cords of EAE mice infected by the recombinant lentivirus. Results were analyzed by one-way ANOVA or Student's t test, as appropriate. Knockdown of the endogenous lncRNA Gm13568 remarkably inhibits the Notch1 expression, astrocytosis, and the phosphorylation of signal transducer and activator of transcription 3 (p-STAT3) as well as the production of inflammatory cytokines and chemokines (IL-6, TNF-α, IP-10) in IL-9-activated astrocytes, in which Gm13568 associates with the transcriptional co-activators CBP/P300 which are enriched in the promoter of Notch1 genes. More importantly, inhibiting Gm13568 with lentiviral vector in astrocytes ameliorates significantly inflammation and demyelination in EAE mice, therefore delaying the EAE process. These findings uncover that Gm13568 regulates the production of inflammatory cytokines in active astrocytes and affects the pathogenesis of EAE through the Notch1/STAT3 pathway. LncRNA Gm13568 may be a promising target for treating MS and demyelinating diseases.


'As long as it helps somebody': why vulnerable people participate in research.
In the past, ethics committees and researchers have avoided research among vulnerable groups because of prevailing perceptions that such research is unethical and difficult. Taking an opposite stand, this article will argue that it is in fact unethical not to research vulnerable populations because, by excluding potential participants from research endeavours, they are being deprived of the benefits to be gained from research. Albeit well meaning, people in vulnerable populations are also being subjected to the paternalistic attitudes of those who believe they know what is in the best interests of others. There is ample evidence in existing literature that, not only is research among vulnerable populations unlikely to result in harm, but there are often benefits to be gained by both participants and researchers. Apart from the generic benefits of increased knowledge and improved health interventions, specific benefits for participants might be: therapeutic, cathartic, educational, empowering, altruistic and social. In contrast, the risks associated with researching vulnerable populations appear to be minimal. Yet, there is still evidence that health care professionals and other gatekeepers continue to hinder research amongst vulnerable populations through unilateral decision-making. This article will review existing literature on the topic of research amongst vulnerable populations, as well as contribute to the argument from the author's own study on a sensitive topic.


Electrocyclic ring opening of charged cis-bicyclo[3.2.0]heptadiene and heterocyclic derivatives. The anti-Woodward-Hoffmann quest (II).
The ring opening reactions of fused cyclobutenes have been the subject of mechanistic debate for decades. Some reports have been published recently suggesting that, in some heterocyclic derivatives, the disrotatory anti-Woodward-Hoffmann mechanism might be responsible for the ring opening. We hereby show that the conrotatory pathway is still the lowest energy alternative for all cases examined, including push-pull substituted 2-thia-4-azabicyclo[3.2.0]hepta-3,6-dienes. Actually, we found that the disrotatory transition state exchanges roles with a double-bond isomerization depending on the substituents around the bicyclic structure.


Nucleotide Binding Preference of the Monofunctional Platinum Anticancer-Agent Phenanthriplatin.
The monofunctional platinum anticancer agent phenanthriplatin generates covalent adducts with the purine bases guanine and adenine. Preferential nucleotide binding was investigated by using a polymerase stop assay and linear DNA amplification with a 163-base pair DNA double helix. Similarly to cisplatin, phenanthriplatin forms the majority of adducts at guanosine residues, but significant differences in both the number and position of platination sites emerge when comparing results for the two complexes. Notably, the monofunctional complex generates a greater number of polymerase-halting lesions at adenosine residues than does cisplatin. Studies with 9-methyladenine reveal that, under abiological conditions, phenanthriplatin binds to the N(1) or N(7) position of 9-methyladenine in approximately equimolar amounts. By contrast, comparable reactions with 9-methylguanine afforded only the N(7) -bound species. Both of the 9-methyladenine linkage isomers (N(1) and N(7) ) exist as two diastereomeric species, arising from hindered rotation of the aromatic ligands about their respective platinum-nitrogen bonds. Eyring analysis of rate constants extracted from variable-temperature NMR spectroscopic data revealed that the activation energies for ligand rotation in the N(1) -bound platinum complex and the N(7) -linkage isomers are comparable. Finally, a kinetic analysis indicated that phenanthriplatin reacts more rapidly, by a factor of eight, with 9-methylguanine than with 9-methyladenine, suggesting that the distribution of lesions formed on double-stranded DNA is kinetically controlled. In addition, implications for the potent anticancer activity of phenanthriplatin are discussed herein.


Profiling the scent of weathered training aids for blood-detection dogs.
At outdoor crime scenes, cadaver-detection and blood-detection dogs may be tasked with locating blood that is days, weeks or months old. Although it is known that the odour profile of blood will change during this time, it is currently unknown how the profile changes when exposed to the environment. Such variables must be studied in order to understand when the odour profile is no longer detectable by the scent-detection dogs and other crime scene tools should be implemented. In this study, blood was deposited onto concrete and varnished wood surfaces and weathered in an outdoor environment over a three-month period. Headspace samples were collected using solid phase microextraction (SPME) and analysed using comprehensive two-dimensional gas chromatography - time-of-flight mass spectrometry (GC×GC-TOFMS). The chemical odour profiles were compared with the behavioural responses of cadaver-detection and blood-detection dogs during training. Data interpretation using principal component analysis (PCA) and hierarchical cluster analysis (HCA) established that the blood odour could no longer be detected using SPME-GC×GC-TOFMS after two months of weathering on both surfaces. Conversely, the blood-detection dogs had difficulty locating the blood samples after one month of weathering on concrete and after one week of weathering on varnished wood. The scent-detection dogs evaluated herein had not been previously exposed to environmentally weathered blood samples during training. Given that this study was conducted to test the dogs' baseline abilities, it is expected that with repeated exposure, the dogs' capabilities would likely improve. The knowledge gained from this study can assist in providing law enforcement with more accurate training aids for blood-detection dogs and can improve their efficiency when deployed to outdoor crime scenes.


Bladder Exposure to Gardnerella Activates Host Pathways Necessary for Escherichia coli Recurrent UTI.
Recurrent urinary tract infections (rUTI) are a costly clinical problem affecting millions of women worldwide each year. The majority of rUTI cases are caused by uropathogenic Escherichia coli (UPEC). Data from humans and mouse models indicate that some instances of rUTI are caused by UPEC emerging from latent reservoirs in the bladder. Women with vaginal dysbiosis, typically characterized by high levels of Gardnerella and other anaerobes, are at increased risk of UTI. Multiple studies have detected Gardnerella in urine collected by transurethral catheterization (to limit vaginal contamination), suggesting that some women experience routine urinary tract exposures. We recently reported that inoculation of Gardnerella into the bladder triggers rUTI from UPEC bladder reservoirs in a mouse model. Here we performed whole bladder RNA-seq to identify host pathways involved in Gardnerella-induced rUTI. We identified a variety host pathways differentially expressed in whole bladders following Gardnerella exposure, such as pathways involved in inflammation/immunity and epithelial turnover. At the gene level, we identified upregulation of Immediate Early (IE) genes, which are induced in various cell types shortly following stimuli like infection and inflammation. One such upregulated IE gene was the orphan nuclear receptor Nur77 (aka Nr4a1). Pilot experiments in Nur77-/- mice suggest that Nur77 is necessary for Gardnerella exposure to trigger rUTI from UPEC reservoirs. These findings demonstrate that bladder gene expression can be impacted by short-lived exposures to urogenital bacteria and warrant future examination of responses in distinct cell types, such as with single cell transcriptomic technologies. The biological validation studies in Nur77-/- mice lay the groundwork for future studies investigating Nur77 and the Immediate Early response in rUTI.


Central Airway Collapse, an Underappreciated Cause of Respiratory Morbidity.
Dyspnea, cough, sputum production, and recurrent respiratory infections are frequently encountered clinical concerns leading patients to seek medical care. It is not unusual for a well-defined etiology to remain elusive or for the therapeutics of a presumed etiology to be incompletely effective. Either scenario should prompt consideration of central airway pathology as a contributor to clinical manifestations. Over the past decade, recognition of dynamic central airway collapse during respiration associated with multiple respiratory symptoms has become more commonly appreciated. Expiratory central airway collapse may represent the answer to this diagnostic void. Expiratory central airway collapse is an underdiagnosed disorder that can coexist with and mimic asthma, chronic obstructive pulmonary disease, and bronchiectasis. Awareness of expiratory central airway collapse and its spectrum of symptoms is paramount to its recognition. This review includes clear definitions, diagnostics, and therapeutics for this challenging condition. We performed a narrative review through the PubMed (MEDLINE) database using the following MeSH terms: airway collapse, tracheobronchomalacia, tracheomalacia, and bronchomalacia. We include reports from systematic reviews, narrative reviews, clinical trials, and observational studies from 2005 to 2020. Two reviewers evaluated potential references. No systematic reviews were found. A total of 28 references were included into our review. Included studies report experience in the diagnosis and/or treatment of dynamic central airway collapse; case reports and non-English or non-Spanish studies were excluded. We describe the current diagnostic dilemma, highlighting the role of dynamic bronchoscopy and tracheobronchial stent trial; outline the complex therapeutic options (eg, tracheobronchoplasty); and present future directions and challenges.


Ethyl 3-oxo-2-(2-phenyl-hydrazinyl-idene)butano-ate: a re-determination.
The previous crystallographic studies [Wang et al. (2005 ▶). Huaxue Yanjiu16, 29-32; Wang et al. (2007 ▶). Youji Huaxue, 27, 524] of the title compound, C(12)H(14)N(2)O(3), gave only the unit-cell dimensions and an R factor with no other details available: the full structure is presented here. The eth-oxy group is disordered over two orientations with refined occupancies of 0.642 (15):0.358 (15). The nine C atoms and two N atoms of the 1-phenyl-2-(propan-2-yl-idene)hydrazine segment of the mol-ecule are close to being coplanar, with a maximum deviation of 0.0779 (14) Å for the phenyl-amino N atom and an intra-molecular N-H⋯O hydrogen bond generates an S(6) ring. In the crystal, pairs of C-H⋯O hydrogen bonds link mol-ecules into inverson dimers, generating R(2) (2)(16) loops.


Aseptic tibial debonding as a cause of early failure in a modern total knee arthroplasty design.
We observed isolated tibial component debonding from the cement in one modern primary TKA design (NexGen LPS 3° tibial tray; Zimmer, Warsaw, IN, USA). This failure mechanism is sparsely reported in the literature. We (1) assessed survivorship of this tibial tray with special emphasis on debonding; (2) described clinical and radiographic features associated with tibial failure; and (3) compared patient and radiographic features of the failures with a matched cohort. A total of 1337 primary TKAs were performed with a cemented NexGen LPS 3° tibial tray over an 11-year period. Twenty-five knees (1.9%) were revised for tibial debonding. BMI and radiographic alignment in the tibial debonding group were compared with a matched control group. Implant survivorship was assessed using tibial debonding as the end point. Survival free of revision from tibial debonding was 100% at 1 year and 97.8% at 5 years. The tibial failures shared a typical radiographic pattern with debonding at the cement-implant interface and subsidence into varus and flexion. We found no link between limb alignment or individual component alignment and failure because 22 of the 25 failures occurred in well-aligned knees. Our standardized followup of patients undergoing TKA at routine intervals allowed us to discover a higher rate of revision resulting from tibial debonding. We have discontinued the use of this particular tibial tray for primary TKA and surveillance for patients undergoing TKA continues to be warranted.


Effect of a monoclonal antibody to PCSK9 on LDL cholesterol.
Proprotein convertase subtilisin/kexin 9 (PCSK9), one of the serine proteases, binds to low-density lipoprotein (LDL) receptors, leading to their accelerated degradation and to increased LDL cholesterol levels. We report three phase 1 studies of a monoclonal antibody to PCSK9 designated as REGN727/SAR236553 (REGN727). In healthy volunteers, we performed two randomized, single ascending-dose studies of REGN727 administered either intravenously (40 subjects) or subcutaneously (32 subjects), as compared with placebo. These studies were followed by a randomized, placebo-controlled, multiple-dose trial in adults with heterozygous familial hypercholesterolemia who were receiving atorvastatin (21 subjects) and those with nonfamilial hypercholesterolemia who were receiving treatment with atorvastatin (30 subjects) (baseline LDL cholesterol, >100 mg per deciliter [2.6 mmol per liter]) or a modified diet alone (10 subjects) (baseline LDL cholesterol, >130 mg per deciliter [3.4 mmol per liter]). REGN727 doses of 50, 100, or 150 mg were administered subcutaneously on days 1, 29, and 43. The primary outcome for all studies was the occurrence of adverse events. The principal secondary outcome was the effect of REGN727 on the lipid profile. Among subjects receiving REGN727, there were no discontinuations because of adverse events. REGN727 significantly lowered LDL cholesterol levels in all the studies. In the multiple-dose study, REGN727 doses of 50, 100, and 150 mg reduced measured LDL cholesterol levels in the combined atorvastatin-treated populations to 77.5 mg per deciliter (2.00 mmol per liter), 61.3 mg per deciliter (1.59 mmol per liter), and 53.8 mg per deciliter (1.39 mmol per liter), for a difference in the change from baseline of -39.2, -53.7, and -61.0 percentage points, respectively, as compared with placebo (P<0.001 for all comparisons). In three phase 1 trials, a monoclonal antibody to PCSK9 significantly reduced LDL cholesterol levels in healthy volunteers and in subjects with familial or nonfamilial hypercholesterolemia. (Funded by Regeneron Pharmaceuticals and Sanofi; ClinicalTrials.gov numbers, NCT01026597, NCT01074372, and NCT01161082.).