Regressive evolution of an effector following a host jump in the Irish potato famine pathogen lineage.
In order to infect a new host species, the pathogen must evolve to enhance infection and transmission in the novel environment. Although we often think of evolution as a process of accumulation, it is also a process of loss. Here, we document an example of regressive evolution of an effector activity in the Irish potato famine pathogen (Phytophthora infestans) lineage, providing evidence that a key sequence motif in the effector PexRD54 has degenerated following a host jump. We began by looking at PexRD54 and PexRD54-like sequences from across Phytophthora species. We found that PexRD54 emerged in the common ancestor of Phytophthora clade 1b and 1c species, and further sequence analysis showed that a key functional motif, the C-terminal ATG8-interacting motif (AIM), was also acquired at this point in the lineage. A closer analysis showed that the P. mirabilis PexRD54 (PmPexRD54) AIM is atypical, the otherwise-conserved central residue mutated from a glutamate to a lysine. We aimed to determine whether this PmPexRD54 AIM polymorphism represented an adaptation to the Mirabilis jalapa host environment. We began by characterizing the M. jalapa ATG8 family, finding that they have a unique evolutionary history compared to previously characterized ATG8s. Then, using co-immunoprecipitation and isothermal titration calorimetry assays, we showed that both full-length PmPexRD54 and the PmPexRD54 AIM peptide bind weakly to the M. jalapa ATG8s. Through a combination of binding assays and structural modelling, we showed that the identity of the residue at the position of the PmPexRD54 AIM polymorphism can underpin high-affinity binding to plant ATG8s. Finally, we conclude that the functionality of the PexRD54 AIM was lost in the P. mirabilis lineage, perhaps owing to as-yet-unknown selection pressure on this effector in the new host environment.


Molecular Rationale for Improved Dynamic Nuclear Polarization of Biomembranes.
Dynamic nuclear polarization (DNP) enhanced solid-state NMR can provide orders of magnitude in signal enhancement. One of the most important aspects of obtaining efficient DNP enhancements is the optimization of the paramagnetic polarization agents used. To date, the most utilized polarization agents are nitroxide biradicals. However, the efficiency of these polarization agents is diminished when used with samples other than small molecule model compounds. We recently demonstrated the effectiveness of nitroxide labeled lipids as polarization agents for lipids and a membrane embedded peptide. Here, we systematically characterize, via electron paramagnetic (EPR), the dynamics of and the dipolar couplings between nitroxide labeled lipids under conditions relevant to DNP applications. Complemented by DNP enhanced solid-state NMR measurements at 600 MHz/395 GHz, a molecular rationale for the efficiency of nitroxide labeled lipids as DNP polarization agents is developed. Specifically, optimal DNP enhancements are obtained when the nitroxide moiety is attached to the lipid choline headgroup and local nitroxide concentrations yield an average e(-)-e(-) dipolar coupling of 47 MHz. On the basis of these measurements, we propose a framework for development of DNP polarization agents optimal for membrane protein structure determination.


Sonic hedgehog (Shh)/Gli modulates the spatial organization of neuroepithelial cell proliferation in the developing chick optic tectum.
Sonic hedgehog (Shh)/Gli pathway plays an important regulatory role on the neuroepithelial cells (NEc) proliferation in the dorsal regions of the developing vertebrate Central Nervous System. The aim of this paper was to analyze the effect of the Shh/Gli signaling pathway activation on the proliferation dynamics and/or the spatial organization of the NEc proliferation activity during early stages of the developing chick optic tectum (OT). In ovo pharmacological gain and loss of hedgehog function approaches were complemented with in vivo electroporation experiments in order to create ectopic sources of either Shh or Gli activator (GliA) proteins in the OT. NEc proliferating activity was analyzed at ED 4/4.5 by recording the spatial co-ordinates of the entire population of mitotic NEc (mNEc) located along OT dorsal-ventral sections. Several space signals (numerical sequences) were derived from the mNEc spatial co-ordinate records and analyzed by different standardized non-linear methods of signal analysis. In ovo pharmacologic treatment with cyclopamine resulted in dramatic failure in the OT expansion while the agonist purmorphamine produced the opposite result, a huge expansion of the OT vesicle. Besides, GliA and Shh misexpressions interfere with the formation of the intertectal fissure located along the dorsal midline. This morphogenetic alteration is accompanied by an increase in the mNEc density. There is a gradient in the response of NEcs to Shh and GliA: the increase in mNEc density is maximal near the dorsal regions and decrease towards the OT-tegmental boundary. Biomathematical analyses of the signals derived from the mNEc records show that both Shh and GliA electroporations change the proliferation dynamics and the spatial organization of the mNEc as revealed by the changes in the scaling index estimated by these methods. The present results show that the Shh/Gli signaling pathway plays a critical role in the OT expansion and modelling. This effect is probably mediated by a differential mitogenic effect that increases the NEc proliferation and modulates the spatial organization of the NEc proliferation activity.


Safety evaluation of genetically modified mustard (V4) seeds in terms of allergenicity: comparison with native crop.
Genetically modified (GM) mustard line (V4) with increased carotenoid content was compared with native mustard to find the difference in allergenic potential, if any. Simulated gastric fluid (SGF) digestibility of crude protein extract from GM as well as its native counterpart mustard crop was envisaged to understand the intended or unintended changes in GM crop along with IgE immunoblotting. BALB/c mice were used as model for allergenicity studies for monitoring total and specific IgE, specific IgG1, histamine level, histopathology, and systemic anaphylaxis score. Allergenicity of mustard was checked in humans by clinical history, skin prick test and IgE levels. Similar results were evident by significant increase in total IgE, specific IgE, IgG1, histamine levels, in GM and native mustard in comparison to control group. Prominent anaphylactic symptoms (score 2: 60%; score 3: 20%; score 4: 20% in native mustard and score 2: 40%; score 3: 40%; score 4: 20% in GM mustard) and eruptive histopathological changes were observed in both GM and native mustard when compared with controls. One protein of approximately 16 kDa was found stable up to 1 h in both GM as well as non GM mustard. IgE immunoblotting detected three protein components of approximately 29, 24 and 16 kDa in both GM and non GM varieties. Collectively, our data demonstrate substantially equivalent allergic responses against GM as well as its native counterpart. Therefore, the GM mustard may be as safe as its native counterpart with reference to allergenic responses.


Financial stress and strain associated with terminal cancer--a review of the evidence.
Financial circumstances are a significant influence on the quality of life for older people and may be important to health and wellbeing at the end of life. The aim of this study is to review the evidence for the existence and consequences of financial stress and strain at the end of life for people dying with cancer. We conducted a systematic search of four electronic databases for studies, providing data on illness-related financial burden (stress), or perception of financial hardship (strain), from patients with terminal cancer or their caregivers. Twenty-four papers were identified from 21 studies published in English between 1980 and 2006, the majority (14) of cross-sectional design. Financial stress was reported in all 13 studies from the USA (median 33%, range 10-66%), but only four sought measures of financial strain. In the USA, specific social consequences, such as moving house or change in employment to cope with caregiving, were reported in four of these studies; one of these also noted changes in treatment choices and avoidance of care for other family members. In studies from outside the USA, there is a dearth of data on financial stresses and the consequences of this for the household, despite widespread reporting of financial strain. To fill a gap in our understanding and improve holistic palliative care, researchers need to ask the questions about the consequences of financial stresses and strain for the health and wellbeing of the household.


Current Trends in Management of Oral Mucositis in Cancer Treatment
Oral Mucositis (OM) is among the most common and dreaded toxicities of cancer therapy. It occurs in almost all patients who receive radiation therapy in which areas of oral and oropharyngeal mucosa are included in the treatment field. With the advent of chemotherapy in 1940 and its extended clinical legacy, it is only within the past two decade or so that mucositis' complex pathobiology has become fully appreciated. There are still many unanswered questions about the risk factors for developing OM, but historically, risk factors have been attributed to both therapy and patient m characteristics. One thing that has been consistent from the initial descriptions of its clinical manifestations has been the frustration on the part of clinicians and patients with the scarcity of therapeutic options to prevent or treat the condition, or effectively ameliorate the symptoms. Clinicians, researchers and those involved in oral and periodontal medicine should join hand in hand in persuit of understanding and developing treatment strategies for treatment of inflammatory conditions like OM in oncology. This will lead to development of effective treatments and reducing the burden of OM and other inflammatory conditions in oncology.


Conversion by Peyer's patch lymphocytes of human enterocytes into M cells that transport bacteria.
The epithelium that lines the gut is impermeable to macromolecules and microorganisms, except in Peyer's patches (PPs), where the lymphoid follicle-associated epithelium (FAE) contains M cells that transport antigens and microorganisms. A cultured system that reproduces the main characteristics of FAE and M cells was established by cultivation of PP lymphocytes with the differentiated human intestinal cell line Caco-2. Lymphocytes settled into the epithelial monolayer, inducing reorganization of the brush border and a temperature-dependent transport of particles and Vibrio cholerae. This model system could prove useful for intestinal physiology, vaccine research, and drug delivery studies.


Flowering responses to altered expression of phytochrome in mutants and transgenic lines of Arabidopsis thaliana (L.) Heynh.
The long-day plant Arabidopsis thaliana (L.) Heynh. flowers early in response to brief end-of-day (EOD) exposures to far-red light (FR) following a fluorescent short day of 8 h. FR promotion of flowering was nullified by subsequent brief red light (R) EOD exposure, indicating phytochrome involvement. The EOD response to R or FR is a robust measure of phytochrome action. Along with their wild-type (WT) parents, mutants deficient in either phytochrome A or B responded similarly to the EOD treatments. Thus, neither phytochrome A nor B exclusively regulated flowering, although phytochrome B controlled hypocotyl elongation. Perhaps a third phytochrome species is important for the EOD responses of the mutants and/or their flowering is regulated by the amount of the FR-absorbing form of phytochrome, irrespective of the phytochrome species. Overexpression of phytochrome A or phytochrome B resulted in differing photoperiod and EOD responses among the genotypes. The day-neutral overexpressor of phytochrome A had an EOD response similar to all of the mutants and WTs, whereas R EOD exposure promoted flowering in the overexpressor of phytochrome B and FR EOD exposure inhibited this promotion. The comparisons between relative flowering times and leaf numbers at flowering of the over-expressors and their WTs were not consistent across photoperiods and light treatments, although both phytochromes A and B contributed to regulating flowering of the transgenic plants.


Extended Neviaser portal approach to antegrade humeral nailing.
Certain arthropathies can distort the normal acromiohumeral relationship and make traditional anterolateral access to the proximal humerus for nailing difficult or impossible. This article presents a case of bilateral antegrade humeral nailing in which the Neviaser portal approach was used for humeral shaft fractures in a patient with distorted shoulder anatomy secondary to severe cuff tear arthropathy and rheumatoid arthritis. Based on a literature review, extending the traditional superomedial (Neviaser) portal to the shoulder to perform humeral nailing has never been described clinically. An 85-year-old woman with rheumatoid arthritis and bilateral cuff tear arthropathy presented after a mechanical fall from standing height with bilateral acute humeral shaft fractures. Preoperative fluoroscopy confirmed the inability to access the traditional starting point with an anterolateral approach due to a shield acromion resulting from cuff tear arthropathy and rheumatoid arthritis. Bilateral locked antegrade humeral nails were successfully placed through a 3-cm incision just off the medial border of the acromion and directly posterior to the acromioclavicular joint (the extended Neviaser portal approach). Postoperatively, the patient demonstrated early evidence of clinical and radiographic union. She was able to return to her preinjury function level, with an active range of motion comparable with her baseline. The Neviaser portal approach to antegrade humeral nailing is an effective solution to diaphyseal humeral fractures when access to the traditional anterolateral proximal humeral starting port is not possible due to distorted shoulder anatomy.


Sequence features accurately predict genome-wide MeCP2 binding in vivo.
Methyl-CpG binding protein 2 (MeCP2) is critical for proper brain development and expressed at near-histone levels in neurons, but the mechanism of its genomic localization remains poorly understood. Using high-resolution MeCP2-binding data, we show that DNA sequence features alone can predict binding with 88% accuracy. Integrating MeCP2 binding and DNA methylation in a probabilistic graphical model, we demonstrate that previously reported genome-wide association with methylation is in part due to MeCP2's affinity to GC-rich chromatin, a result replicated using published data. Furthermore, MeCP2 co-localizes with nucleosomes. Finally, MeCP2 binding downstream of promoters correlates with increased expression in Mecp2-deficient neurons.


Operating Room Performance Improves after Proficiency-Based Virtual Reality Cataract Surgery Training.
To investigate the effect of virtual reality proficiency-based training on actual cataract surgery performance. The secondary purpose of the study was to define which surgeons benefit from virtual reality training. Multicenter masked clinical trial. Eighteen cataract surgeons with different levels of experience. Cataract surgical training on a virtual reality simulator (EyeSi) until a proficiency-based test was passed. Technical performance in the operating room (OR) assessed by 3 independent, masked raters using a previously validated task-specific assessment tool for cataract surgery (Objective Structured Assessment of Cataract Surgical Skill). Three surgeries before and 3 surgeries after the virtual reality training were video-recorded, anonymized, and presented to the raters in random order. Novices (non-independently operating surgeons) and surgeons having performed fewer than 75 independent cataract surgeries showed significant improvements in the OR-32% and 38%, respectively-after virtual reality training (P = 0.008 and P = 0.018). More experienced cataract surgeons did not benefit from simulator training. The reliability of the assessments was high with a generalizability coefficient of 0.92 and 0.86 before and after the virtual reality training, respectively. Clinically relevant cataract surgical skills can be improved by proficiency-based training on a virtual reality simulator. Novices as well as surgeons with an intermediate level of experience showed improvement in OR performance score.


Robust regression with asymmetric heavy-tail noise distributions.
In the presence of a heavy-tail noise distribution, regression becomes much more difficult. Traditional robust regression methods assume that the noise distribution is symmetric, and they downweight the influence of so-called outliers. When the noise distribution is asymmetric, these methods yield biased regression estimators. Motivated by data-mining problems for the insurance industry, we propose a new approach to robust regression tailored to deal with asymmetric noise distribution. The main idea is to learn most of the parameters of the model using conditional quantile estimators (which are biased but robust estimators of the regression) and to learn a few remaining parameters to combine and correct these estimators, to minimize the average squared error in an unbiased way. Theoretical analysis and experiments show the clear advantages of the approach. Results are on artificial data as well as insurance data, using both linear and neural network predictors.


Reactive molecular dynamics simulations of hydration shells surrounding spherical TiO2 nanoparticles: implications for proton-transfer reactions.
In many potential applications, nanoparticles are typically in an aqueous medium. This has strong influence on the stability, optical properties and reactivity, in particular for their functionalization. Therefore, the understanding of the chemistry at the interface between the solvent and the nanoparticle is of utmost importance. In this work, we present a comparative ReaxFF reactive molecular dynamics investigation on spherical TiO2 nanoparticles (NSs) of realistic size, with diameters from 2.2 to 4.4 nm, immersed in a large drop of bulk water. After force field validation for its use for a curved anatase TiO2 surface/water interface, we performed several simulations of the TiO2 nanoparticles of increasing size in a water drop. We found that water can be adsorbed jointly in a molecular and dissociative way on the surface. A Langmuir isotherm indicating an adsorption/desorption mechanism of water on the NS is observed. Regarding the dissociative adsorption, atomistic details reveal two different mechanisms, depending on the water concentration around the NS. At low coverage, the first mechanism involves direct dissociation of a single water molecule, whereas, at higher water coverage, the second mechanism is a proton transfer reaction involving two water molecules, also known as Grotthuss-like mechanism. Thermal annealing simulations show that several water molecules remain on the surface in agreement with the experimental reports. The capacity of adsorption is higher for the 2.2 and 3.0 nm NSs than for the 4.4 nm NS. Finally, a comparative investigation with flat surfaces indicates that NSs present a higher water adsorption capacity (undissociated and dissociated) than flat surfaces, which can be rationalized considering that NSs present many more low-coordinated Ti atoms available for water adsorption.


Impact of replacing cytology with human papillomavirus testing for cervical cancer screening on the prevalence of Trichomonas vaginalis: a modelling study.
Trichomonas vaginalis (TV) is the most common curable STI worldwide and is associated with increased risk of HIV acquisition and serious reproductive morbidities. The prevalence of TV infection is very low in Australian cities, and this is thought to be at least partly due to incidental detection and treatment of TV in women participating in the cervical cytology screening programme. In 2017, the national cervical screening programme will transition to a new model based on testing for high-risk (HR) human papillomavirus (HPV), with a reduced frequency and commencement at an older age. We model the potential impact of this transition on TV prevalence in Australia. A mathematical model was developed to describe the transmission of TV in the general population and used to evaluate scenarios that capture the switch from cytology-based screening to HR HPV testing. Under these scenarios, individuals with asymptomatic TV who test negative for HR HPV will remain undiagnosed and untreated. We estimate the change in TV prevalence expected to occur due to the switch from cytology to HR HPV testing and changes to the frequency and age at commencement of screening. Our results suggest that with the transition to HR HPV testing, TV prevalence may increase from the current ~0.4% to 2.8% within 20 years if TV testing coverage is not increased and HR HPV prevalence does not decline further. If HR HPV prevalence continues to decline at its current rate with ongoing vaccination, TV prevalence is predicted to increase to 3.0% within this time frame. Our modelling suggests that in a setting like Australia, where TV can be detected incidentally through cytology-based cervical screening, a transition to HPV testing is likely to result in increasing TV prevalence over time unless additional measures are implemented to increase TV testing and treatment.


The clementine mission to the moon: scientific overview.
In the course of 71 days in lunar orbit, from 19 February to 3 May 1994, the Clementine spacecraft acquired just under two million digital images of the moon at visible and infrared wavelengths. These data are enabling the global mapping of the rock types of the lunar crust and the first detailed investigation of the geology of the lunar polar regions and the lunar far side. In addition, laser-ranging measurements provided the first view of the global topographic figure of the moon. The topography of many ancient impact basins has been measured, and a global map of the thickness of the lunar crust has been derived from the topography and gravity.


Protein-metal ion interactions, stoichiometries and relative affinities determined by on-line size exclusion gel filtration mass spectrometry.
The modulation of metal ions on protein function is well recognized and of paramount importance in protein biochemistry. To date, very few methods allow direct determination of protein-metal ion interactions, as well as exact stoichiometric binding ratios. In this work we demonstrate the usefulness of two on-line size exclusion gel filtration mass spectrometry approaches to directly detect protein-metal ion adducts, as well as determine exact protein-metal ion stoichiometries. We show that on-line size exclusion column chromatography (SEC) and rapid in-line desalting (RILED) coupled to microelectrospray mass spectrometry (microESI-MS) can be used for such analyses. The SEC approach can be effectively used to both separate proteins in a complex mixture and exchange buffers prior to the electrospray process. While RILED does not allow for protein separation, it provides a much faster high-throughput desalting procedure than the conventional SEC technique. Specifically, we show that SEC/microESI-MS and RILED/MS can be used to determine calcium ion binding stoichiometries to a high-affinity, metal ion binding protein, calbindin D(28K). Furthermore, the same approaches can also be used to determine metal ion binding stoichiometries of low-affinity metal-binding proteins such as Spo0F.


The SWI/SNF chromatin remodeling subunit BAF57 is a critical regulator of estrogen receptor function in breast cancer cells.
Estrogen receptors (ERs) play critical roles in both normal mammary gland development and in the formation and progression of breast tumors, constituting a major therapeutic target for breast cancer treatment. We have previously described that ER transcriptional activity is potentiated by BAF57, a core subunit of the mammalian SWI/SNF chromatin remodeling complex. Here we provide evidence demonstrating an important role for BAF57 as regulator of ER functions in breast cancer cells. Different experimental manipulations leading to the abrogation of BAF57 expression and/or function severely reduced the expression of various endogenous ER target genes and blocked estrogen-stimulated proliferation in ZR-75-1 breast cancer cells. Moreover, using a structure-function analysis, we have defined the protein domains required for the functional interaction between ERalpha and BAF57, including a key region within the hinge of ERalpha that is essential for BAF57 recruitment and its function on ER-mediated transcription. Interestingly, we found that BAF57 is an ER subtype-selective modulator that specifically regulates ERalpha-mediated transcription. Taken together, our results suggest that targeting BAF57 could represent a new way to effectively inhibit the action of ERalpha.


A novel role for vitamin K1 in a tyrosine phosphorylation cascade during chick embryogenesis.
The development of the embryo is dependent upon a highly coordinated repertoire of cell division, differentiation, and migration. Protein-tyrosine phosphorylation plays a pivotal role in the regulation of these processes. Vitamin K-dependent gamma-carboxylated proteins have been identified as ligands for a unique family (Tyro 3 and 7) of receptor tyrosine kinases (RTKs) with transforming ability. The involvement of vitamin K metabolism and function in two well characterized birth defects, warfarin embryopathy and vitamin K epoxide reductase deficiency, suggests that developmental signals from K-dependent pathways may be required for normal embryogenesis. Using a chick embryogenesis model, we now demonstrate the existence of a vitamin K1-dependent protein-tyrosine phosphorylation cascade involving c-Eyk, a member of the Tyro 12 family, and key intracellular proteins, including focal adhesion kinase (pp125FAK), paxillin, and pp60src. This cascade is sensitive to alteration in levels or metabolism of vitamin K1. These findings provide a major clue as to why, in the mammalian (and human) fetus, the K-dependent proteins are maintained in an undercarboxylated state, even to the point of placing the newborn at hemorrhagic risk. The precise regulation of vitamin K1-dependent regulatory pathways would appear to be critical for orderly embryogenesis.


Increased development of radiographic hip osteoarthritis in individuals with high bone mass: a prospective cohort study.
Individuals with high bone mass (HBM) have a greater odds of prevalent radiographic hip osteoarthritis (OA), reflecting an association with bone-forming OA sub-phenotypes (e.g. osteophytosis, subchondral sclerosis). As the role of bone mineral density (BMD) in hip OA progression is unclear, we aimed to determine if individuals with HBM have increased incidence and/or progression of bone-forming OA sub-phenotypes. We analysed an adult cohort with and without HBM (L1 and/or total hip BMD Z-score > + 3.2) with pelvic radiographs collected at baseline and 8-year follow-up. Sub-phenotypes were graded using the OARSI atlas. Superior/inferior acetabular/femoral osteophyte and medial/superior joint space narrowing (JSN) grades were summed and Δosteophyte and ΔJSN derived. Pain and functional limitations were quantified using the WOMAC questionnaire. Associations between HBM status and change in OA sub-phenotypes were determined using multivariable linear/logistic regression, adjusting for age, sex, height, total body fat mass, follow-up time and baseline sub-phenotype grade. Generalised estimating equations accounted for individual-level clustering. Of 136 individuals, 62% had HBM at baseline, 72% were female and mean (SD) age was 59 (10) years. HBM was positively associated with both Δosteophytes and ΔJSN (adjusted mean grade differences between individuals with and without HBM βosteophyte = 0.30 [0.01, 0.58], p = 0.019 and βJSN = 0.10 [0.01, 0.18], p = 0.019). Incident subchondral sclerosis was rare. HBM individuals had higher WOMAC hip functional limitation scores (β = 8.3 [0.7, 15.98], p = 0.032). HBM is associated with the worsening of hip osteophytes and JSN over an average of 8 years, as well as increased hip pain and functional limitation.


Methylation of Ir(iii)-tetrazolato complexes: an effective route to modulate the emission outputs and to switch to antimicrobial properties.
Two neutral cyclometalated Ir(iii)-tetrazolato complexes that differ by variations of the substituents on either the phenylpyridine or the tetrazolate ligand have been converted into the corresponding methylated and cationic analogues. NMR (1H and 13C) characterization of the Ir(iii) complexes provided the results in agreement with the chemo- and regioselective character of methylation at the N-3 position of the Ir(iii)-coordinated tetrazolato ring. This evidence was further corroborated by the analysis of the molecular structures of the cationic complexes obtained by X-ray diffraction. In view of the photophysical properties, the addition of a methyl moiety to neutral Ir(iii) tetrazolates, which behave as sky-blue or orange phosphors, caused a systematic red shift of their phosphorescence output. The transformation of neutral Ir(iii) tetrazolates into cationic Ir(iii)-tetrazole complexes was screened for any eventual antimicrobial activity in vitro against Gram negative (E. coli) and Gram positive (D. radiodurans) microorganisms. While both kinds of complexes were not active against E. coli, the conversion of the neutral Ir(iii) tetrazolates into the corresponding methylated and cationic Ir(iii)tetrazole derivatives determined the turn-on of a good to excellent antimicrobial activity toward Gram positive Deinococcus radiodurans, a non-pathogenic bacterium that is listed as one of the toughest microorganisms in light of its outstanding resistance to radiation and oxidative stress.


HemoHIM, a herbal preparation, alleviates airway inflammation caused by cigarette smoke and lipopolysaccharide.
HemoHIM, herbal preparation has designed for immune system recovery. We investigated the anti-inflammatory effect of HemoHIM on cigarette smoke (CS) and lipopolysaccharide (LPS) induced chronic obstructive pulmonary disease (COPD) mouse model. To induce COPD, C57BL/6 mice were exposed to CS for 1 h per day (eight cigarettes per day) for 4 weeks and intranasally received LPS on day 26. HemoHIM was administrated to mice at a dose of 50 or 100 mg/kg 1h before CS exposure. HemoHIM reduced the inflammatory cell count and levels of tumor necrosis factor receptor (TNF)-α, interleukin (IL)-6 and IL-1β in the broncho-alveolar lavage fluid (BALF) induced by CS+LPS exposure. HemoHIM decreased the inflammatory cell infiltration in the airway and inhibited the expression of iNOS and MMP-9 and phosphorylation of Erk in lung tissue exposed to CS+LPS. In summary, our results indicate that HemoHIM inhibited a reduction in the lung inflammatory response on CS and LPS induced lung inflammation via the Erk pathway. Therefore, we suggest that HemoHIM has the potential to treat pulmonary inflammatory disease such as COPD.


Anti-inflammatory and autonomic effects of electroacupuncture in a rat model of diet-induced obesity.
To study the effect of electroacupuncture (EA) on the cholinergic anti-inflammatory pathway (CAP) by measurement of vagal activity in rats with high-fat diet (HFD)-induced obesity. Diet-induced obesity (DIO) was induced in 30 rats by feeding them a HFD for 12 weeks. A further 10 rats fed normal food comprised the lean diet (LD) control group. DIO rats were further subdivided into three groups that received a HFD only (HFD group, n=10), a HFD plus electroacupuncture (HFD+EA group, n=10) or a HFD plus minimal acupuncture (HFD+MA group, n=10). EA and MA treatments were continued for 8 weeks. Heart rate variability (HRV) was used to measure the function of the autonomic nervous system before and after treatment. ELISA was used to determine acetylcholine (ACh) and tumour necrosis factor (TNF)-α levels in the serum. Real-time PCR was used to assess the mRNA expression of α7-subtype nicotinic acetylcholine cholinergic receptors (α7nAChRs) and TNF-α in the mesenteric white adipose tissues (MWAT). EA but not MA significantly reduced rats' bodyweight. No difference was found in the low frequency (LF), high frequency (HF) and the balance between LF and HF (LF/HF) components of HRV before treatment. After the EA intervention, HF was elevated and LF/HF was reduced in the HFD+EA group comparedwith the HFD group. TNF-α in the serum and MWAT were increased in the HFD group, but were reduced in the HFD+EA group. Furthermore, EA promoted expression of α7nAChRs and ACh in the MWAT. There was no difference between the HFD and HFD+MA groups for any indices. EA enhanced vagal activity, promoted ACh release and activated α7nAChRs in the MWAT, leading to inhibition of proinflammatory cytokine production.


Studies on chemical constituents of Gracilaria verrucosa.
A new sterol glucoside, (24R)-5 alpha-stigmast-9-(11)-en-3beta-D-glucopyranoside (1) has been isolated from a methanol extract of the red alga Gracilaria verrucosa, while cholesterol and (Z)-9-hexadecenoic acid (palmitoleic acid) have been isolated from a dichloromethane-acetone extract of the same alga. Additionally, an oily fraction of a dichloromethane-acetone extract has been investigated by gas chromatography/mass spectrometry (GC/MS). The main components in the oily fraction were heneicosanoic acid (49.8%), (Z)-9-octadecenoic acid (15.8%), and 15-tetracosenoic acid (15.6%) in addition to a few hydrocarbons, simple aromatics and a new compound, namely 3,4-dimethyl-5-carboxyclic acid-1-oxacyclopent-3-en-2-one. The structures of the isolated compounds have been elucidated by spectroscopic methods.


The HAND Database: a gateway to understanding the role of HIV in HIV-associated neurocognitive disorders.
Despite an augmented research effort and scale-up of highly active antiretroviral therapy, a high prevalence of HIV-1-associated neurocognitive disorders (HAND) persists in the HIV-infected population. Nearly 50 % of all HIV-1-infected individuals suffer from a neurocognitive disorder due to neural and synaptodendritic damage. Challenges in HAND research, including limited availability of brain tissue from HIV patients, variation in HAND study protocols, and virus genotyping inconsistency and errors, however, have resulted in studies with insufficient power to delineate molecular mechanisms underlying HAND pathogenesis. There exists, therefore, a great need for a reliable and centralized resource specific to HAND research, particularly for epidemiological study and surveillance in resource-limited countries where severe forms of HAND persist. To address the aforementioned imperative need, here we present the HAND Database, a resource containing well-curated and up-to-date HAND virus information and associated clinical and epidemiological data. This database provides information on 5,783 non-redundant HIV-1 sequences from global HAND research published to date, representing a total of 163 unique individuals that have been assessed for HAND. A user-friendly interface allows for flexible searching, filtering, browsing, and downloading of data. The most comprehensive database of its kind, the HAND Database not only bolsters current HAND research by increasing sampling power and reducing study biases caused by protocol variation and genotyping inconsistency, it allows for comparison between HAND studies across different dimensions. Development of the HAND Database has also revealed significant knowledge gaps in HIV-driven neuropathology. These gaps include inadequate sequencing of viral genes beyond env, lack of HAND viral data from HIV epidemiologically important regions including Asian and Sub-Saharan African countries, and biased sampling toward the male gender, all factors that impede efforts toward providing an improved quality of life to HIV-infected individuals, and toward elimination of viruses in the brain. Our aim with the HAND database is to provide researchers in both the HIV and neuroscience fields a comprehensive and rigorous data source toward better understanding virus compartmentalization and to help in design of improved strategies against HAND viruses. We also expect this resource, which will be updated on a regular basis, to be useful as a reliable reference for further HAND epidemiology studies. The HAND Database is freely available and accessible online at http://www.handdatabase.org .


The Lyt-2 molecule recognizes residues in the class I alpha 3 domain in allogeneic cytotoxic T cell responses.
The involvement of the different domains of the MHC class I molecule in CTL recognition was investigated. mAbs specific for the alpha 1/alpha 2 domains of H-2Ld interfered with both the primary and secondary generation and effector function of in vitro Ld-specific CTL. mAbs specific for the alpha 3 domain of H-2Ld interfered with the generation and function of primary in vitro Ld-specific CTL; however, there was no effect on the in vitro generation of secondary CTL and only partial inhibition of their function. In vivo treatment with graft-specific antibodies to both the alpha 3 domain and the alpha 1/alpha 2 domains together resulted in a dramatic enhancement of Ld- or Dd-disparate skin grafts, whereas the individual mAbs showed minimal effects. This suggested that the class I alpha 3 domain is recognized by alloreactive CTL. Several approaches were undertaken to examine whether recognition of the alpha 3 domain determinants is mediated by the Lyt-2 molecule. When mAbs specific for the alpha 3 domain of either H-2Ld or H-2Dd were used in vivo and in vitro, the resulting CTL population was not inhibited by antibody to the alpha 3 domain and was only partially inhibited by antibody to Lyt-2. We therefore observed a correlation between the effects of antibody to the class I alpha 3 domain of the target molecule and antibody to the Lyt-2 molecule on the CTL. To further test the relationship between CTL recognition of the alpha 3 domain and the involvement of Lyt-2, we used a cell expressing a mutation in the alpha 3 domain of the Dd molecule. The mutation resulted in a single amino acid substitution of glu to lys at residue 227 of the alpha 3 domain. Consistent with an earlier report, cells expressing the mutant Dd lys molecule were not lysed by CTL from a primary stimulation against the wild-type Dd glu molecule. However, this same cell line was killed by the Lyt-2-independent secondary Dd-specific CTL generated in the presence of antibody to the alpha 3 domain in vivo and in vitro. Furthermore, cells expressing the mutant Dd lys molecule failed to stimulate a primary response. In conclusion, several independent lines of evidence indicate that residues in the alpha 3 domain of the class I molecule are involved in recognition by the Lyt-2 molecule, and that Lyt-2-mediated recognition can be specifically blocked using mAb to determinants in the alpha 3 domain.


Forensic analysis of laser printed ink by X-ray fluorescence and laser-excited plume fluorescence.
We demonstrated a minimally destructive two-tier approach for multielement forensic analysis of laser-printed ink. The printed document was first screened using a portable-X-ray fluorescence (XRF) probe. If the results were not conclusive, a laser microprobe was then deployed. The laser probe was based on a two-pulse scheme: the first laser pulse ablated a thin layer of the printed ink; the second laser pulse at 193 nm induced multianalytes in the desorbed ink to fluoresce. We analyzed four brands of black toners. The toners were printed on paper in the form of patches or letters or overprinted on another ink. The XRF probe could sort the four brands if the printed letters were larger than font 20. It could not tell the printing sequence in the case of overprints. The laser probe was more discriminatory; it could sort the toner brands and reveal the overprint sequence regardless of font size while the sampled area was not visibly different from neighboring areas even under the microscope. In terms of general analytical performance, the laser probe featured tens of micrometer lateral resolution and tens to hundreds of nm depth resolution and atto-mole mass detection limits. It could handle samples of arbitrary size and shape and was air compatible, and no sample pretreatment was necessary. It will prove useful whenever high-resolution and high sensitivity 3D elemental mapping is required.


The impact of managed care on patients' trust in medical care and their physicians.
Social trust in health care organizations and interpersonal trust in physicians may be mutually supportive, but they also diverge in important ways. The success of medical care depends most importantly on patients' trust that their physicians are competent, take appropriate responsibility and control, and give their patients' welfare the highest priority. Utilization review and structural arrangements in managed care potentially challenge trust in physicians by restricting choice, contradicting medical decisions and control, and restricting open communication with patients. Gatekeeping and incentives to limit care also raise serious trust issues. We argue that managed care plans rather than physicians should be required to disclose financial arrangements, that limits be placed on incentives that put physicians at financial risk, and that professional norms and public policies should encourage clear separation of interests of physicians from health plan organization and finance.


RIP2 is a novel NF-kappaB-activating and cell death-inducing kinase.
Through specific interactions with members of the tumor necrosis receptor (TNFR) family, adapter molecules such as the serine/threonine (Ser/Thr) kinase RIP mediate divergent signaling pathways including NF-kappaB activation and cell death. In this study, we have identified and characterized a novel 61-kDa protein kinase related to RIP that is a component of both the TNFR-1 and the CD40 signaling complexes. Receptor interacting protein-2 (RIP2) contains an N-terminal domain with homology to Ser/Thr kinases and a C-terminal caspase activation and recruitment domain (CARD), a homophilic interaction motif that mediates the recruitment of caspase death proteases. Overexpression of RIP2 signaled both NF-kappaB activation and cell death. Mutational analysis revealed the pro-apoptotic function of RIP2 to be restricted to its C-terminal CARD domain, whereas the intact molecule was necessary for NF-kappaB activation. RIP2 interacted with other members of the TNFR-1 signaling complex, including inhibitor of apoptosis protein cIAP1 and with members of the TNFR-associated factor (TRAF) family, specifically TRAF1, TRAF5, and TRAF6, but not with TRAF2, TRAF3, or TRAF4. These TRAF interactions mediate the recruitment of RIP2 to receptor signaling complexes.


A Multisite Collaborative to Decrease Inappropriate Antibiotics in Urgent Care Centers.
Urgent care (UC; a convenient site to receive care for ambulatory-sensitive) centers conditions; however, UC clinicians showed the highest rate of inappropriate antibiotic prescriptions among outpatient settings according to national billing data. Antibiotic prescribing practices in pediatric-specific UC centers were not known but assumed to require improvement. The aim of this multisite quality improvement project was to reduce inappropriate antibiotic prescribing practices for 3 target diagnoses in pediatric UC centers by a relative 20% by December 1, 2019. The Society of Pediatric Urgent Care invited pediatric UC clinicians to participate in a multisite quality improvement study from June 2019 to December 2019. The diagnoses included acute otitis media (AOM), otitis media with effusion, and pharyngitis. Algorithms based on published guidelines were used to identify inappropriate antibiotic prescriptions according to indication, agent, and duration. Sites completed multiple intervention cycles from a menu of publicly available antibiotic stewardship materials. Participants submitted data electronically. The outcome measure was the percentage of inappropriate antibiotic prescriptions for the target diagnoses. Process measures were use of delayed antibiotics for AOM and inappropriate testing in pharyngitis. From 20 UC centers, 157 providers submitted data from 3833 encounters during the intervention cycles. Overall inappropriate antibiotic prescription rates decreased by a relative 53.9%. Inappropriate antibiotic prescribing decreased from 57.0% to 36.6% for AOM, 54.6% to 48.4% for otitis media with effusion, and 66.9% to 11.7% for pharyngitis. Participating pediatric UC providers decreased inappropriate antibiotic prescriptions from 60.3% to 27.8% using publicly available interventions.


Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community.
Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene segments was used to profile microbial populations inhabiting different temperature regions in the microbial mat community of Octopus Spring, Yellowstone National Park. DGGE allowed a rapid evaluation of the distributions of amplifiable sequence types. Profiles were essentially identical within regions of the mat defined by one temperature range but varied between sites with different temperature ranges. Individual DGGE bands were sequenced, and the sequences were compared with those previously obtained from the mat by cloning and from cultivated Octopus Spring isolates. Two known cyanobacterial populations and one known green nonsulfur bacterium-like population were detected by DGGE, as were many new cyanobacterial and green nonsulfur and green sulfur bacterium-like populations and a novel bacterial population of uncertain phylogenetic affiliation. The distributions of several cyanobacterial populations compared favorably with results obtained previously by oligonucleotide probe analyses and suggest that adaptation to temperature has occurred among cyanobacteria which are phylogenetically very similar.


Platelet-derived growth factor receptor beta-mediated phosphorylation of MUC1 enhances invasiveness in pancreatic adenocarcinoma cells.
MUC1 is a heterodimeric transmembrane glycoprotein that is overexpressed and aberrantly glycosylated in ductal adenocarcinomas. Differential phosphorylation of the MUC1 cytoplasmic tail (MUC1CT) has been associated with signaling events that influence the proliferation and metastasis of cancer cells. We identified a novel tyrosine phosphorylation site (HGRYVPP) in the MUC1CT by mass spectrometric analysis of MUC1 from human pancreatic adenocarcinoma cell lines. Analyses in vitro and in vivo showed that platelet-derived growth factor receptor beta (PDGFRbeta) catalyzed phosphorylation of this site and of tyrosine in the RDTYHPM site. Stimulation of S2-013.MUC1F cells with PDGF-BB increased nuclear colocalization of MUC1CT and beta-catenin. PDGF-BB stimulation had no significant effect on cell proliferation rate; however, it enhanced invasion in vitro through Matrigel and in vivo tumor growth and metastases. Invasive properties of the cells were significantly altered on expression of phosphorylation-abrogating or phosphorylation-mimicking mutations at these sites. We propose that interactions of MUC1 and PDGFRbeta induce signal transduction events that influence the metastatic properties of pancreatic adenocarcinoma.


An autoradiographic study of the distribution of binding sites for the novel alpha7-selective nicotinic radioligand [3H]-methyllycaconitine in the mouse brain.
[3H]-Methyllycaconitine ([3H]-MLA) is a new radioligand with selectivity for alpha7-type neuronal nicotinic acetylcholine receptors (nAChRs). In our previous study [Davies, A.R.L., Hardick, D.J., Blagbrough, I.S., Potter, B.V.L., Wolstenholme, A.J. & Wonnacott, S. (1999) Neuropharmacology, 38, 679-690], this radioligand labelled a single class of site in rat brain membranes; its pharmacology and distribution in crudely dissected brain regions closely paralleled that of the well-established alpha7-ligand [125I]-alpha-bungarotoxin. However, a small population of [3H]-MLA binding sites was apparently insensitive to alpha-bungarotoxin. Here we have extended the study to mouse brain, using autoradiography to examine the distribution of [3H]-MLA and [125I]-alpha-bungarotoxin binding sites. [3H]-MLA labelled a single class of site in mouse brain membranes with a KD of 2.2 nM and a Bmax of 45.6 fmol/mg protein. Specific binding, defined by unlabelled MLA (Ki = 0.69 nM), was completely inhibited by (-)-nicotine (Ki = 1.62 microM), whereas alpha-bungarotoxin inhibited only 85% of specific binding (Ki = 3.5 nM). The distributions of [125I]-alpha-bungarotoxin and [3H]-MLA binding sites were compared by autoradiography, and binding was quantitated in 72 brain regions. Binding of both radioligands was highly correlated, with highest densities in the dorsal tegmental nucleus of the pons, colliculi and hippocampus. Serial sections labelled with [3H]-MLA in the absence or presence of unlabelled MLA or alpha-bungarotoxin provided no evidence for any alpha-bungarotoxin-resistant binding. The results are discussed in terms of binding sites that are inaccessible to alpha-bungarotoxin in membrane preparations. This study demonstrates the utility of [3H]-MLA for characterization of alpha7-type nicotinic receptors in mammalian brain, and suggests that it labels a population identical to that defined by [125I]-alpha-bungarotoxin.


Phase II study of twice-daily high-dose thoracic radiotherapy alternating with cisplatin and vindesine for unresectable stage III non-small-cell lung cancer: Japan Clinical Oncology Group Study 9306.
To evaluate the efficacy and toxicity of high-dose thoracic radiotherapy (TRT) alternating with chemotherapy (CH) for unresectable stage III non--small-cell lung cancer (NSCLC). Forty-one patients received TRT with 1.5 Gy twice daily, 5 days a week, on weeks 1, 2, 5, 6, and 9, up to a total dose of 66 to 72 Gy, alternating with cisplatin 80 mg/m(2) on day 1 and vindesine 3 mg/m(2) on days 1 and 8, repeated every 4 weeks, for two or three courses beginning on week 3. The median (range) total dose of TRT and number of CH courses were 72 Gy (16.5 to 72 Gy) and three (zero to three), respectively. Delay in TRT > or = 5 days was observed in 24 (75%) of 32 patients who completed the projected treatment, due to leukopenia in 12, esophagitis in seven, infection in two, and other causes in three patients. Partial responses were obtained in 36 patients (88%). The median survival time and 3- and 5-year survival rates were 18.4 months, 24%, and 10%, respectively. Grade 3 or 4 leukopenia and esophagitis developed in 32 and seven patients, respectively. Grade 3 or 4 late esophageal toxicity developed in two patients. Alternating high-dose TRT and CH for stage III NSCLC produced a high response rate with median and long-term survival comparable to prior trials utilizing standard approaches in this population. Acute and late esophageal toxicity was observed and interruption of TRT was required in most of the patients.


How dangerousness evolves after court-ordered compulsory psychiatric admission: explorative prospective observational cohort study.
Compulsory admission is commonly regarded as necessary and justified for patients whose psychiatric condition represents a severe danger to themselves and others. However, while studies on compulsory admissions have reported on various clinical and social outcomes, little research has focused specifically on dangerousness, which in many countries is the core reason for compulsory admission.AimsTo study changes in dangerousness over time in adult psychiatric patients admitted by compulsory court order, and to relate these changes to these patients' demographic and clinical characteristics. In this explorative prospective observational cohort study of adult psychiatric patients admitted by compulsory court order, demographic and clinical data were collected at baseline. At baseline and at 6 and 12 month follow-up, dangerousness was assessed using the Dangerousness Inventory, an instrument based on the eight types of dangerousness towards self or others specified in Dutch legislation on compulsory admissions. We used descriptive statistics and logistic regression to analyse the data. We included 174 participants with a court-ordered compulsory admission. At baseline, the most common dangerousness criterion was inability to cope in society. Any type of severe or very severe dangerousness decreased from 86.2% at baseline to 36.2% at 6 months and to 28.7% at 12 months. Being homeless at baseline was the only variable which was significantly associated with persistently high levels of dangerousness. Dangerousness decreased in about two-thirds of the patients after court-ordered compulsory admission. It persisted, however, in a substantial minority (approximately one-third).Declaration of interestNone.


Proximal femoral nail shows better concordance of gait analysis between operated and uninjured limbs compared to hemiarthroplasty in intertrochanteric femoral fractures.
The purpose of this study was to compare the results of pedobarographic gait analysis between the patients treated by proximal femoral nail or bipolar partial hemiarthroplasty due to intertrochanteric fractures. Thirty-seven patients with a minimum 1-year follow-up who had been operated for intertrochanteric fractures were evaluated clinically, radiologically and with pedobarographic gait analysis. Proximal femoral nail had been performed to 21 patients (group A), whilst 16 patients had been operated by partial bipolar hemiarthroplasty (group B). Pedobarographic analysis was performed by measuring plantar pressure, force and contact area values in both static and dynamic manner. Pedobarographic results of operated limb were compared among groups. Same data's also were compared between operated and uninjured limbs in each group to determine any asymmetry on weight-bearing. Average follow-up period in group A and group B was 36 (12-56) and 30 (12-48) months, respectively. There were no statistically significant differences among groups in terms of age, gender, body mass index, type and side of fracture, follow-up period, leg length discrepancy and postoperative hip scores. When the pedobarographic results of operated limb were compared, group B showed much more plantar force and pressure values than group A, on both static and dynamic evaluations. If the evaluation was taken into consideration to comparison of pedobarographic results between operated and uninjured limbs in each group, we found asymmetry in static load bearing, caused by higher load on uninjured limb in both groups. However, there was no statistically significant asymmetry between operated and uninjured limbs in respect to dynamic pedobarographic parameters for patients in group A. On the contrary, operated limbs in group B exposed much more plantar force and pressure values than uninjured limbs, which indicated asymmetric weight-bearing on dynamic evaluation. Assessment of pedobarographic parameters can be another way of measuring the results of treatment in intertrochanteric fractures. Uninjured limbs of patients expose much more loading than operated limbs in postoperative static evaluation for both treatment options. However in dynamic evaluation, there is a better concordance of gait analysis between both limbs in patients operated by proximal femoral nail.


Antisense inhibition of acetylcholinesterase gene expression causes transient hematopoietic alterations in vivo.
Hematopoietic acetylcholinesterase (ACHE) gene expression and its implication for development were studied by in vivo administration to mice of an antisense phosphorothioate oligonucleotide targetted toward ACHE (AS-ACHE). Hematopoietic alterations were observed by differential cell counts and ACHE mRNA levels determined by quantified RNA polymerase chain reaction (RNA-PCR) and in situ hybridization analyses. In control mice, injected with phosphate-buffered saline and untreated, ACHE mRNA labeling with ACHE [35S]cRNA was about 10-fold higher on megakaryocytes (MK) compared with all other bone marrow cells and increased by 20-fold during MK development, similar to reports for MK actin mRNA. Drastic reductions occurred in the bone marrow lymphocyte and erythroid fractions 12 days following intraperitoneal injection of AS-ACHE (5 micrograms/g weight) into groups of four mice. RNA-PCR revealed over 1000-fold decreases in ACHE mRNA levels in lymph nodes and bone marrow at this time, while actin mRNA levels dropped by 10 and 100-fold in lymph nodes and bone marrow of AS-ACHE treated mice compared with controls. In view of the developmental increase in MK actin, this suggested arrest in MK development as well. By 20 days postinjection, bone marrow actin mRNA was fully restored and the sensitive in situ hybridization technique revealed that ACHE mRNA levels were also restored and reached levels only 2-3-fold lower than in controls in all bone marrow cells of AS-ACHE treated mice. Moreover, lymphocytes and erythroid cells repopulated to levels 25% above normal, and promegakaryocyte and mature MK fractions of the total MK were 3 and 2-fold higher, respectively, than in controls.(ABSTRACT TRUNCATED AT 250 WORDS)


Variation in Revascularization Practice and Outcomes in Asymptomatic Stable Ischemic Heart Disease.
The aims of this study were to assess variation in revascularization of asymptomatic patients with stable ischemic heart disease, identify the predictors of variation, and determine if it was associated with clinical outcomes. Management of stable ischemic heart disease in asymptomatic patients with obstructive coronary artery disease is controversial, potentially leading to practice variation. A retrospective observational cohort study was performed using population-based data from Ontario, Canada, in patients with asymptomatic stable ischemic heart disease and obstructive coronary artery disease. The cohort was divided on the basis of treatment strategy: revascularization or medical therapy. Hospitals were allocated into tertiles of their revascularization ratio. Outcomes included death and nonfatal myocardial infarction. Hierarchical logistic regression was used to assess the predictors of revascularization, with median odds ratios used to quantify variation. Proportional hazards models were used to determine the association between management strategy and outcomes. The cohort included 9,897 patients, 47% treated with medical therapy and 53% with revascularization. Between hospitals, 2-fold variation existed in the ratio of revascularized to medically treated patients. However, the variation across hospitals was not explained by patient, physician, or hospital factors (median odds ratio in null model: 1.25; median odds ratio in full model: 1.31). Revascularization was associated with a hazard ratio of 0.81 (95% confidence interval: 0.69 to 0.96) for death and a hazard ratio of 0.58 (95% confidence interval: 0.46 to 0.73) for myocardial infarction, with this benefit consistent across tertiles of revascularization ratio. Wide variation was observed in revascularization practice that was not explained by known factors. Despite this variation, a clinical benefit was observed with revascularization that was consistent across hospitals.


Molecular determinants of voltage-dependent human ether-a-go-go related gene (HERG) K+ channel block.
The structural determinants for the voltage-dependent block of ion channels are poorly understood. Here we investigate the voltage-dependent block of wild-type and mutant human ether-a-go-go related gene (HERG) K(+) channels by the antimalarial compound chloroquine. The block of wild-type HERG channels expressed in Xenopus oocytes was enhanced as the membrane potential was progressively depolarized. The IC(50) was 8.4 +/- 0.9 microm when assessed during 4-s voltage clamp pulses to 0 mV. Chloroquine also slowed the apparent rate of HERG deactivation, reflecting the inability of drug-bound channels to close. Mutation to alanine of aromatic residues (Tyr-652 or Phe-656) located in the S6 domain of HERG greatly reduced the potency of channel block by chloroquine (IC(50) > 1 mm at 0 mV). However, mutation of Tyr-652 also altered the voltage dependence of the block. In contrast to wild-type HERG, block of Y652A HERG channels was diminished by progressive membrane depolarization, and complete relief from block was observed at +40 mV. HERG channel block was voltage-independent when the hydroxyl group of Tyr-652 was removed by mutating the residue to Phe. Together these findings indicate a critical role for Tyr-652 in voltage-dependent block of HERG channels. Molecular modeling was used to define energy-minimized dockings of chloroquine to the central cavity of HERG. Our experimental findings and modeling suggest that chloroquine preferentially blocks open HERG channels by cation-pi and pi-stacking interactions with Tyr-652 and Phe-656 of multiple subunits.


Cysteinyl leukotrienes promote human airway smooth muscle migration.
Cysteinyl leukotrienes promote airway smooth muscle (ASM) contraction and proliferation. Little is known about their role in ASM migration. We investigated this using cultured human ASMs (between the second and fifth passages) obtained from the large airways of resected nonasthmatic lung. Platelet-derived growth factor-BB (1 ng/ml) promoted significant (3.5-fold) ASM migration of myocytes across collagen-coated 8- micro m polycarbonate membranes in Transwell culture plates. Leukotriene E(4) (10(-7), 10(-8), 10(-9) M) did not demonstrate a chemotactic effect; it did promote chemokinesis. Priming by leukotriene E(4) (10(-7) M) significantly augmented the directional migratory response to platelet-derived growth factor (1.5-fold, p < 0.05). This was blocked by montelukast (10(-6) M), demonstrating the effect to be mediated by the cysteinyl leukotriene receptor. The "priming effect" was also partially attenuated by prostaglandin E(2) (10(-7) M). Whereas both the chemokinetic and the chemotactic "primed" responses were equally attenuated by a p38 mitogen-activated protein kinase inhibitor (SB203580, 25 micro M) and by a Rho-kinase inhibitor (Y27632, 10 micro M), the chemotactic response showed greater inhibition than chemokinesis by a phosphatidylinositol-3 kinase inhibitor (LY294002, 50 micro M). These experiments suggest that cysteinyl leukotrienes play an augmentary role in human ASM migration. The phosphatidylinositol-3 kinase pathway is a key signaling mechanism in the chemotactic migration of ASM cells in response to cysteinyl leukotrienes.


Experimental analysis of protogyny in Aquilegia canadensis (Ranunculaceae).
Dichogamy is very common in flowering plants and is widely thought to reduce pollen-pistil interference, especially self-pollination. Yet, the functional significance of dichogamy has rarely been studied. We investigated the nature and functioning of dichogamy in eastern Ontario populations of Aquilegia canadensis, a highly selfing columbine previously described as protogynous. We then manipulated flowers to determine whether increased protogyny would reduce self-fertilization. Contrary to previous reports, A. canadensis is not dichogamous. Controlled pollinations in a greenhouse showed that pollen tubes generally begin to develop after anther dehiscence. Although stigmas can collect pollen early in floral development, naturally pollinated flowers collected from four populations had few pollen grains on stigmas and almost no pollen tubes in styles until after anther dehiscence. Limited pollen deposition before anther dehiscence was also associated with low nectar availability and limited sepal expansion. Because inbreeding depression is strong in this species, selection may favor increased protogyny if it reduces selfing. We tested this hypothesis by comparing the level of selfing in flowers rendered protogynous by the removal of the first 19 (of 39) anthers to develop, with nonprotogynous control flowers. Contrary to expectations, protogyny did not reduce selfing. Our results emphasize the importance of detailed field observations and manipulative experiments in understanding the nature and functional significance of dichogamy.


Long-term host unresponsiveness to encapsulated xenogeneic myoblasts after transient immunosuppression.
Encapsulating cells prevents the immune destruction of allogeneic cells in the subcutaneous site as well as allogeneic and xenogeneic cells in the central nervous system. However, when encapsulated xenogeneic cells are implanted s.c., they may be subject to rejection by the host. Murine C2C12 myoblasts engineered to secrete mouse erythropoietin (mEpo) were used to evaluate the response of control versus FK506-treated xenogeneic recipients (Fischer rats) to encapsulated myoblasts implanted in the s.c. site. Encapsulated C2C12 mEpo cells were rapidly eliminated in immunocompetent Fischer rats. Devices transplanted into nude rats induced a sustained increase in the hematocrit, associated with an extended viability of the encapsulated cells. Short-term immunosuppression with FK506, for periods lasting either 1, 2, or 4 weeks after implantation, permitted the long-term survival of encapsulated C2C12 mEpo cells in Fischer rats. Animals increased their hematocrits to more than 70% and maintained these levels for 13 weeks, independent of the duration of FK506 treatment. Unencapsulated C2C12 mEpo cells injected i.m. in immunosuppressed animals were rejected over this same period. Encapsulation alone cannot protect xenogeneic myoblasts from immune destruction in the s.c. site. These results highlight the importance of combining the technique of cell encapsulation with transient immunosuppression to achieve long-term survival of xenografted myoblasts in a peripheral immunoreactive site.


Spectrophotometric methods for the determination of the antidepressant drug paroxetine hydrochloride in tablets.
Simple, sensitive, and accurate visible spectrophotometric methods are described for the determination of paroxetine hydrochloride (PA) in tablets. Among them, the first 3 methods are based on the ion-pair complexes of PA formed with bromothymol blue (BTB), bromophenol blue (BPB), and bromocresol green (BCG) in aqueous acidic buffers. The complex species extracted into chloroform were quantitatively measured at 414 nm with BTB and BCG and at 412 nm with BPB. Beer's law was obeyed over the concentration ranges of 2-20, 2-16, and 2-16 microg/mL, respectively. The fourth method described is based on a coupling reaction between PA and 7-chloro-4-nitrobenzofurazon (NBD-Cl) in borate buffer, pH 8.5, in which a yellow reaction product that was measured at 478 nm was formed. The Beer's law range for this method was 2-10 microg/mL. The last method developed describes the interaction of PA base, as an n-electron donor, with 7,7,8,8-tetracyanoquinodimethane (TCNQ), as a pi-acceptor, in acetonitrile to give blue-colored TCNQ- radical anion with absorption maxima at 750 and 845 nm. Measured at 845 nm, the absorbance-concentration plot was rectilinear over the range of 1.5-15 microg/mL. The new methods developed were successfully applied to the determination of PA in tablets without any interference from common tablet excipients. The results of the methods were in good agreement with those obtained with an official liquid chromatographic method. This report describes first colorimetric methods for the determination of PA.


Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) reduces vascular endothelial growth factor expression in allergen-induced airway inflammation.
Vascular endothelial growth factor (VEGF) plays a pivotal role in the pathogenesis of bronchial asthma. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been implicated in regulating cell survival signaling through the phosphoinositide 3-kinase (PI3K)/Akt pathway. The key role of PI3K in VEGF-mediated signal transduction is established. However, the effects of PTEN on VEGF-mediated signaling in asthma are unknown. This study aimed to determine the effect of PI3K inhibitors and PTEN on VEGF expression in allergen-induced airway inflammation. We have used a female C57BL/6 mouse model for asthma to determine the role of PTEN in allergen-induced airway inflammation, specifically in the expression of VEGF. Allergen-induced airway inflammation leads to increased activity of PI3K in lung tissue. These mice develop the following typical pathophysiological features of asthma in the lungs: increased numbers of inflammatory cells of the airways; airway hyper-responsiveness; increased expression of interleukin (IL)-4, IL-5, IL-13, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, regulated on activation normal T cell expressed and secreted (RANTES), and eotaxin; increased vascular permeability; and increased levels of VEGF. Administration of PI3K inhibitors or adenoviruses carrying PTEN cDNA reduced the symptoms of asthma and decreased the increased levels of plasma extravasation and VEGF in allergen-induced asthmatic lungs. These results indicate that PTEN reduces VEGF expression in allergen-induced airway inflammation.


Hope predicts positive functional role outcomes in acute rehabilitation populations.
The purpose of this study was to examine the association between facilitating psychological variables and functional rehabilitation outcomes following acute medical rehabilitation. Using a longitudinal design and correlational and regression analyses, we studied 174 adults who were participating in inpatient rehabilitation for acute spinal cord dysfunction, stroke, amputation, or orthopedic surgery recovery. All participants completed the Hope Scale, Positive and Negative Affect Schedule, and Functional Independence Measure (FIM) during the first days of their inpatient stay, and then were contacted 3 months after discharge to complete the Craig Hospital Assessment and Reporting Technique (CHART) and FIM. Hope accounted for a statistically significant amount of the variance in the prediction of functional role participation at 3 months postdischarge (as measured by the CHART) above and beyond the variance accounted for by demographic and severity variables. In contrast, positive affect was not found to contribute to the prediction of functional role participation, and neither hope nor positive affect contributed to the prediction of functional skill level (FIM). The results indicate that positive psychological variables present during the rehabilitation stay, such as hopefulness, may contribute to the prediction of functional outcomes after discharge in rehabilitation populations. These findings suggest that incorporating interventions that enhance hope and build on the individual's psychological strengths may be useful to improve participation outcomes following acute medical rehabilitation.


pH effects on the electrochemical reduction of CO(2) towards C2 products on stepped copper.
We present a microkinetic model for CO(2) reduction (CO(2)R) on Cu(211) towards C2 products, based on energetics estimated from an explicit solvent model. We show that the differences in both Tafel slopes and pH dependence for C1 vs C2 activity arise from differences in their multi-step mechanisms. We find the depletion in C2 products observed at high overpotential and high pH to arise from the 2nd order dependence of C-C coupling on CO coverage, which decreases due to competition from the C1 pathway. We further demonstrate that CO(2) reduction at a fixed pH yield similar activities, due to the facile kinetics for CO2 reduction to CO on Cu, which suggests C2 products to be favored for CO2R under alkaline conditions. The mechanistic insights of this work elucidate how reaction conditions can lead to significant enhancements in selectivity and activity towards higher value C2 products.


Autologous and allogeneic stem cell transplantation in adult ALL: the Swedish Adult ALL Group experience.
Adult patients with acute lymphoblastic leukaemia (ALL) have been treated according to national protocols in Sweden since 1986. Stem cell transplantation (SCT) has been recommended in first remission for patients with risk factors for relapse, and for standard risk patients only after relapse. In this retrospective study, the results of autologous and allogeneic SCT in these populations were evaluated. In total, 187 patients with a median age of 34 years (17-66 years) underwent SCT. The 5-year disease-free survival (DFS), for all patients, was 26% (Confidence intervals (CI) 20-32%). The 5-year DFS was higher for patients transplanted in first remission 32% (CI 24-40%) compared to 14% (CI 5-23%; P<0.0001) in patients transplanted beyond first remission. No significant differences in DFS (P=0.06) were determined between autologous, related donor and unrelated donor SCT in the whole cohort. A lower relapse rate was counterbalanced by higher treatment-related mortality in patients undergoing allogeneic SCT. In Philadelphia-positive ALL, allogeneic SCT was superior to autologous SCT, with a 5-year DFS of 30% (CI 12-47%) vs 0% (P=0.04). Limited chronic graft-versus-host-disease (GVHD) was associated with an improved DFS of 53% (CI 38-69%) compared to no chronic GVHD of 22% (CI 10-36%; P=0.0008), indicating a clinically important graft-versus-leukaemia effect.


Chimeric human immunodeficiency virus type 1 and feline immunodeficiency virus reverse transcriptases: role of the subunits in resistance/sensitivity to non-nucleoside reverse transcriptase inhibitors.
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are specific for human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and do not inhibit HIV-2. Given that the amino acids lining the NNRTI-specific pocket of HIV-1 RT display higher similarity to the corresponding feline immunodeficiency virus (FIV) RT amino acids than to HIV-2 RT, the susceptibility of FIV RT and chimeric HIV-1/FIV RTs to NNRTIs and the role of the p51 subunit in the inhibitory action of NNRTIs were investigated. We found that the wild-type FIV RT and the FIVp66/HIVp51 chimeric enzyme showed no susceptibility for NNRTIs. On the other hand, the chimeric HIVp66/FIVp51 RT retained a sensitivity spectrum for NNRTIs similar to that of the wild-type HIV-1 RT. The noncompetitive nature of inhibition of HIV-1 RT by nevirapine was also observed with the HIVp66/FIVp51 chimeric enzyme. Inhibition of the chimeric RTs by nucleoside reverse transcriptase inhibitors and foscarnet was in the same range as observed for the corresponding HIVp66/HIVp51 and FIVp66/FIVp51 wild-type enzymes. The chimeric RTs had an affinity (K(m)) for their dNTP substrate and template/primer comparable with that of the wild-type HIV-1 and FIV RTs, but their catalytic efficacy (k(cat)) was markedly decreased. This decreased catalytic efficacy of the RT chimeras may suggest suboptimal interactions between p66 and p51 in the chimeric enzymes. Our results point to a minor role of the p51 subunit in the sensitivity to RT inhibitors.


Estradiol up-regulates antiapoptotic Bcl-2 messenger ribonucleic acid and protein in tumorigenic ovarian surface epithelium cells.
Most epithelial ovarian tumors appear to arise from the ovarian surface epithelium (OSE). Even though it has been suggested that estrogen may be associated with ovarian tumorigenesis, the exact role of estrogen in the regulation of apoptosis in neoplastic OSE cells remains uncertain. Immortalized OSE (IOSE) cell lines were generated from human normal OSE. These cell lines represent early neoplastic (IOSE-29), tumorigenic (IOSE-29EC), and late neoplastic (IOSE-29EC/T4 and IOSE-29EC/T5) transformation stages from human normal OSE. The present studies demonstrated that both mRNAs and proteins of estrogen receptor (ER) alpha and beta were expressed in IOSE cell lines. No difference was observed in normal OSE and IOSE-29 cells, whereas treatment with 17beta-estradiol (E(2); 10(-8)-10(-6) M) resulted in an increased thymidine incorporation and DNA content per culture in IOSE-29EC cells. This effect of E(2) was attenuated with tamoxifen treatment (10(-6) M), the estrogen antagonist, suggesting that the effect of E(2) is mediated through specific ERs. There was no stimulatory effect on thymidine incorporation before day 6, but after 6 days of E(2) treatment, thymidine incorporation was significantly increased. Because the ratio of thymidine incorporation to DNA content per culture did not change, this E(2) effect does not appear to indicate stimulation of proliferation but, rather, inhibition of apoptosis. In addition, treatment with tamoxifen (10(-6) M) induced apoptosis up to 3-fold in IOSE-29EC cells, whereas cotreatment with E(2) (10(-8)-10(-6) M) plus tamoxifen attenuated tamoxifen-induced apoptosis in a dose-dependent manner. Both proapoptotic bax and antiapoptotic bcl-2 at messenger RNA (mRNA) and protein levels were expressed in IOSE cell lines. Interestingly, treatments with E(2) resulted in a significant increase of bcl-2 mRNA and protein levels (2- and 1.7-fold, respectively), whereas no difference was observed in bax mRNA level. Thus, E(2) may enhance survival of IOSE-29EC by up-regulating bcl-2, and antiapoptotic bcl-2 may be a dominant regulator of apoptotic pathway in these cells. In conclusion, the present study indicates that early neoplastic (IOSE-29), tumorigenic (IOSE-29EC), and late neoplastic (IOSE-29EC/T4 and T5) OSE cells expressed both ERalpha and ERbeta at the mRNA and protein levels. In addition, E(2) prevented tamoxifen induced-apoptosis through ERs. The mechanism of E(2) action may be associated with up-regulation of bcl-2 gene at mRNA and protein levels. These results suggest that estrogen may play a role in ovarian tumorigenesis by preventing apoptosis in tumorigenic OSE cells.


Uncertainty and the value of diagnostic information, with application to axillary lymph node dissection in breast cancer.
In clinical decision making, it is common to ask whether, and how much, a diagnostic procedure is contributing to subsequent treatment decisions. Statistically, quantification of the value of the information provided by a diagnostic procedure can be carried out using decision trees with multiple decision points, representing both the diagnostic test and the subsequent treatments that may depend on the test's results. This article investigates probabilistic sensitivity analysis approaches for exploring and communicating parameter uncertainty in such decision trees. Complexities arise because uncertainty about a model's inputs determines uncertainty about optimal decisions at all decision nodes of a tree. We present the expected utility solution strategy for multistage decision problems in the presence of uncertainty on input parameters, propose a set of graphical displays and summarization tools for probabilistic sensitivity analysis in multistage decision trees, and provide an application to axillary lymph node dissection in breast cancer.


Can OCT be sensitive to nanoscale structural alterations in biological tissue?
Exploration of nanoscale tissue structures is crucial in understanding biological processes. Although novel optical microscopy methods have been developed to probe cellular features beyond the diffraction limit, nanometer-scale quantification remains still inaccessible for in situ tissue. Here we demonstrate that, without actually resolving specific geometrical feature, OCT can be sensitive to tissue structural properties at the nanometer length scale. The statistical mass-density distribution in tissue is quantified by its autocorrelation function modeled by the Whittle-Mateŕn functional family. By measuring the wavelength-dependent backscattering coefficient μb(λ) and the scattering coefficient μs, we introduce a technique called inverse spectroscopic OCT (ISOCT) to quantify the mass-density correlation function. We find that the length scale of sensitivity of ISOCT ranges from ~30 to ~450 nm. Although these sub-diffractional length scales are below the spatial resolution of OCT and therefore not resolvable, they are nonetheless detectable. The sub-diffractional sensitivity is validated by 1) numerical simulations; 2) tissue phantom studies; and 3) ex vivo colon tissue measurements cross-validated by scanning electron microscopy (SEM). Finally, the 3D imaging capability of ISOCT is demonstrated with ex vivo rat buccal and human colon samples.


Proficiency program for real-time PCR diagnosis of Bordetella pertussis infections in French hospital laboratories and at the French National Reference Center for Whooping Cough and other Bordetelloses.
With the support of a ministerial program for innovative and expensive technologies, dedicated to the economic evaluation of laboratory diagnosis of pertussis by real-time PCR, external quality assessment for real-time IS481 PCR was carried out. Coordinated by the National Centre of Reference of Pertussis and other Bordetelloses (NCR), this study aimed to harmonize and to assess the performances of eight participating microbiology hospital laboratories throughout the French territory. Between January 2006 and February 2007, 10 proficiency panels were sent by the NCR (ascending proficiency program), representing a total of 49 samples and including eight panels to analyze and evaluate the global sensitivity and specificity of real-time PCR, one to assess the limit of detection, and one to evaluate nucleic acid extraction methods. As part of the descending proficiency program, extracted DNA from clinical samples was sent by the eight participating laboratories in different panels and analyzed by the NCR. In the ascending proficiency analysis, the sensitivity and specificity of the real-time PCR methods were 92.2% and 94.3%, respectively. The limit of detection of the different methods ranged between 0.1 and 1 fg/microl (0.2 to 2 CFU/microl). The nucleic acid extraction methods showed similar performances. During the descending proficiency analysis, performed with 126 samples, the result of the NCR for 15 samples (11.9%) was discordant with the result obtained by the source laboratory. Despite several initial differences, harmonization was easy and performances were homogeneous. However, the risk of false-positive results remains quite high, and we strongly recommend establishment of uniform quality control procedures performed regularly.


Resting cerebral blood flow: a potential biomarker of the effects of HIV in the brain.
HIV enters the brain soon after infection causing neuronal damage and microglial/astrocyte dysfunction leading to neuropsychological impairment. We examined the impact of HIV on resting cerebral blood flow (rCBF) within the lenticular nuclei (LN) and visual cortex (VC). This cross-sectional study used arterial spin labeling MRI (ASL-MRI) to measure rCBF within 33 HIV+ and 26 HIV- subjects. Nonparametric Wilcoxon rank sum test assessed rCBF differences due to HIV serostatus. Classification and regression tree (CART) analysis determined optimal rCBF cutoffs for differentiating HIV serostatus. The effects of neuropsychological impairment and infection duration on rCBF were evaluated. rCBF within the LN and VC were significantly reduced for HIV+ compared to HIV- subjects. A 2-tiered CART approach using either LN rCBF < or =50.09 mL/100 mL/min or LN rCBF >50.09 mL/100 mL/min but VC rCBF < or =37.05 mL/100 mL/min yielded an 88% (29/33) sensitivity and an 88% (23/26) specificity for differentiating by HIV serostatus. HIV+ subjects, including neuropsychologically unimpaired, had reduced rCBF within the LN (p = 0.02) and VC (p = 0.001) compared to HIV- controls. A temporal progression of brain involvement occurred with LN rCBF significantly reduced for both acute/early (<1 year of seroconversion) and chronic HIV-infected subjects, whereas rCBF in the VC was diminished for only chronic HIV-infected subjects. Resting cerebral blood flow (rCBF) using arterial spin labeling MRI has the potential to be a noninvasive neuroimaging biomarker for assessing HIV in the brain. rCBF reductions that occur soon after seroconversion possibly reflect neuronal or vascular injury among HIV+ individuals not yet expressing neuropsychological impairment.


The Hog1 MAPK prevents cross talk between the HOG and pheromone response MAPK pathways in Saccharomyces cerevisiae.
The MAPKKK Ste11p functions in three Saccharomyces cerevisiae MAPK cascades [the high osmolarity glycerol (HOG), pheromone response, and pseudohyphal/invasive growth pathways], but its activation in response to high osmolarity stimulates only the HOG pathway. To determine what restricts cross-activation of MAPK cascades (cross talk), we have studied mutants in which the pheromone response pathway is activated by high osmolarity (1 M sorbitol). We found that mutations in the HOG1 gene, encoding the p38-type MAPK of the HOG pathway, and in the PBS2 gene, encoding the activating kinase for Hog1p, allowed osmolarity-induced activation of the pheromone response pathway. This cross talk required the osmosensor Sho1p, as well as Ste20p, Ste50p, the pheromone response MAPK cascade (Ste11p, Ste7p, and Fus3p or Kss1p), and Ste12p but not Ste4p or the MAPK scaffold protein, Ste5p. The cross talk in hog1 mutants induced multiple responses of the pheromone response pathway: induction of a FUS1::lacZ reporter, morphological changes, and mating in ste4 and ste5 mutants. We suggest that Hog1p may prevent osmolarity-induced cross talk by inhibiting Sho1p, perhaps as part of a feedback control on the HOG pathway. We have also shown that Ste20p and Ste50p function in the Sho1p branch of the HOG pathway and that a second osmosensor in addition to Sho1p may activate Ste11p. Finally, we have found that pseudohyphal growth exhibited by wild-type (HOG1) strains depends on SHO1, suggesting that Sho1p may be a receptor that feeds into the pseudohyphal growth pathway.


Investigation of Sacral Needle Depth in Minimally Invasive Sacrocolpopexy.
The aims of the study were to optimize surgical safety and to minimize vertebral disc puncture during sacral needle placement at the time of minimally invasive sacrocolpopexy. Cadaveric studies report that the anterior longitudinal ligament (ALL), which covers the vertebral disc and vertebrae, has a reported thickness of only 1.4 to 2.3 mm at L5-S1. Intervertebral disc puncture can accelerate disc degeneration, disc herniation, and loss of disc height, a risk that may be avoidable. After institutional review board approval, research consent was obtained from women undergoing primary laparoscopic sacrocolpopexy. Intraoperatively, sacral sutures were placed in the ALL with a 1.5 cm diameter CV-2 needle using Gore-Tex suture. Depth measurements were collected using a laparoscopic ultrasound transducer positioned on the sacral promontory (SP) between the 2 ends of the needle visible through the ALL. Two still-frame US images of the single needle were taken using the BK Medical software. Needle depth was calculated by measuring the distance from the top of the ALL to the needle. Two satisfactory intraoperative images were obtained for all 9 participants. The mean needle depth at the SP was 3.96 mm. The interpatient needle depth varied from 2.07 to 9.04 mm. In most participants (78%), the sacral needle depth exceeded 2.3 mm, suggesting that there may be risk to sacral suture placement without depth guidance at the promontory. During minimally invasive sacrocolpopexy, the depth of the ALL and the placement of the needle at the SP may result in inadvertent disc penetration. Surgeons should be conscious of the minimal depth of the ALL and consider placing the suture below the promontory to avoid the disc.


Characterization of a plant (rice) translin and its comparative analysis with human translin.
For the first time, a plant (rice) translin was characterized. The rice translin protein, which was octameric in native state, bound efficiently to single-stranded DNA and RNA. Translin, a DNA-/RNA-binding protein, is expressed in brain, testis and in certain malignancies. It is involved in chromosomal translocation, mRNA metabolism, transcriptional regulation and telomere protection. Studies from human, mice, drosophila and yeast have revealed that it forms an octameric ring, which is important for its function. In spite of the absence of neuronal functions and cancer processes, translin is present in plant systems, but information on plant translin is lacking. Here we report the characterization of a plant (rice) translin. Translin cDNA from O. sativa was cloned into an expression vector; protein was over-expressed in E. coli and subsequently purified to homogeneity. Circular dichroism and homology-based modeling showed that the rice translin protein was similar to the other translin proteins. Native PAGE and gel-filtration analyses showed rice translin to form an octamer and this octameric assembly was independent of disulphide bonds. Rice translin bound to single-stranded DNA sequences like human translin, but not to the double-stranded DNA. Rice translin bound more efficiently to linear DNA (with staggered ends) than open or closed circular DNA. Rice translin also bound to RNA, like its human counterpart. Rice translin displays all the characteristic properties of the translin group of proteins and does indeed qualify as a bonafide "translin" protein. To our knowledge, this is the first report wherein the translin protein from a plant source has been functionally characterized. Understanding the translin biology from plant systems will give the new insights into its functional role during plant development.


Cloud Products from the Earth Polychromatic Imaging Camera (EPIC): Algorithms and Initial Evaluation.
This paper presents the physical basis of the EPIC cloud product algorithms and an initial evaluation of their performance. Since June 2015, EPIC has been providing observations of the sunlit side of the Earth with its 10 spectral channels ranging from the UV to the near-IR. A suite of algorithms has been developed to generate the standard EPIC Level 2 Cloud Products that include cloud mask, cloud effective pressure/height, and cloud optical thickness. The EPIC cloud mask adopts the threshold method and utilizes multichannel observations and ratios as tests. Cloud effective pressure/height is derived with observations from the O2 A-band (780 nm and 764 nm), and B-band (680 nm and 688 nm) pairs. The EPIC cloud optical thickness retrieval adopts a single channel approach where the 780 nm and 680 nm channels are used for retrievals over ocean and over land, respectively. Comparison with co-located cloud retrievals from geosynchronous earth orbit (GEO) and low earth orbit (LEO) satellites shows that the EPIC cloud product algorithms are performing well and are consistent with theoretical expectations. These products are publicly available at the Atmospheric Science Data Center at the NASA Langley Research Center for climate studies and for generating other geophysical products that require cloud properties as input.


A model of glucose-insulin homeostasis in man that incorporates the heterogeneous fast pool theory of pancreatic insulin release.
Current physiologic knowledge about glucose-insulin homeostasis in liver, brain, pancreas, kidney, peripheral tissues, and central vascular organs has been synthesized to form a whole-system mathematical model of glucose metabolism in normal, ideal man. In addition to data of other workers, results from more than 100 intravenous glucose tolerance tests, including variable dosage, variable duration of infusion, and double pulse studies, were used to determine model structure and parameters. Model and clinical testing have focused particularly on the fast phase of insulin response to vascular glucose. The model incorporates blood circulation and equilibration of substances between vascular and interstitial spaces, and it assumes constant fractional clearance of insulin by liver and kidney. Studies using a double pulse of glucose suggest that the time derivative of glucose level is not the sole or predominant influence on fast phase insulin release, but that preinfusion glucose level and/or previous glucose exposure of the pancreas are also important. Variable dosage glucose studies suggest that the amount of insulin released during the fast phase rather than the insulin release rate is regulated by the glucose level. A two-pool, heterogeneous threshold mechanism for beta cell response to glucose is presented that is compatible with the clinical results.


Short tandem repeat number estimation from paired-end reads for multiple individuals by considering coalescent tree.
Two types of approaches are mainly considered for the repeat number estimation in short tandem repeat (STR) regions from high-throughput sequencing data: approaches directly counting repeat patterns included in sequence reads spanning the region and approaches based on detecting the difference between the insert size inferred from aligned paired-end reads and the actual insert size. Although the accuracy of repeat numbers estimated with the former approaches is high, the size of target STR regions is limited to the length of sequence reads. On the other hand, the latter approaches can handle STR regions longer than the length of sequence reads. However, repeat numbers estimated with the latter approaches is less accurate than those with the former approaches. We proposed a new statistical model named coalescentSTR that estimates repeat numbers from paired-end read distances for multiple individuals simultaneously by connecting the read generative model for each individual with their genealogy. In the model, the genealogy is represented by handling coalescent trees as hidden variables, and the summation of the hidden variables is taken on coalescent trees sampled based on phased genotypes located around a target STR region with Markov chain Monte Carlo. In the sampled coalescent trees, repeat number information from insert size data is propagated, and more accurate estimation of repeat numbers is expected for STR regions longer than the length of sequence reads. For finding the repeat numbers maximizing the likelihood of the model on the estimation of repeat numbers, we proposed a state-of-the-art belief propagation algorithm on sampled coalescent trees. We verified the effectiveness of the proposed approach from the comparison with existing methods by using simulation datasets and real whole genome and whole exome data for HapMap individuals analyzed in the 1000 Genomes Project.


Retroviral transfer of the recombinant human erythropoietin receptor gene into single hematopoietic stem/progenitor cells from human cord blood increases the number of erythropoietin-dependent erythroid colonies.
To test whether an enforced expression of a lineage-specific cytokine receptor would influence the proliferation/differentiation of hematopoietic stem/progenitor cells, retroviral vectors containing the human erythropoietin receptor (hEpoR) gene were used to transduce the hEpoR gene into phenotypically sorted subsets of cells. CD34 , CD34++CD33-, and CD34++CD33+ populations of human cord blood, highly enriched for hematopoietic stem/progenitor cells, were sorted and plated as single cells per well in methylcellulose culture medium containing early acting growth factors in the presence or absence of Epo. The hEpoR gene was efficiently transduced into single high proliferative potential colony-forming cells (HPP-CFC) and multipotential (colony-forming unit granulocyte, erythroid, monocyte, megakaryocyte [CFU-GEMM]), erythroid (burst-forming unit-erythroid [BFU-E]), and granulocyte-macrophage (colony-forming unit-granulocyte-macrophage [CFU-GM]) progenitor cells. As expected in cultures grown in the absence of Epo, no BFU-E or CFU-GEMM colonies grew. In the presence of Epo, the hEpoR-gene transduced cells formed significantly more CFU-GEMM and BFU-E colonies than did the controls. A significant decrease in HPP-CFC colonies was also observed under these conditions. Little or no effect of hEpoR gene transduction was apparent in the numbers of CFU-GM colonies formed in the presence or absence of Epo. All of the above results were similar whether the cell populations assessed were CD34 or their CD33- or CD33+ subsets plated in the presence of growth factors at 200 cells/mL or after limiting dilution at 2 cells/well. These results suggest that the profile of detectable stem/progenitors can be altered by retrovirus-mediated expression of the hEpoR gene.


Cloning and nucleotide sequence of the carboxynorspermidine decarboxylase gene from Vibrio alginolyticus.
The gene (nspC) encoding carboxynorspermidine decarboxylase (CANS DC), the last enzyme in norspermidine biosynthesis, in Vibrio alginolyticus was isolated by immuno-screening and its complete nucleotide sequence was determined. Sequence analysis of the subcloned fragment (2.0 kb) revealed an ORF of 1131 bp encoding a protein of 377 amino acids with a calculated molecular mass of 42,008 Da. The sequence of 20 N-terminal amino acids of purified CANS DC was found to be identical to that predicted from the nspC gene. A putative ribosome binding sequence was observed 8 bp upstream from the translation start site (ATG), and promoter- and terminator-like sequences were detected upstream and downstream of the ORF, respectively. Database searches identified no similar proteins, but the deduced amino acid sequence contained a putative pyridoxal 5'-phosphate binding region similar to those of the bacterial meso-2,6-diaminopimelate decarboxylases and eukaryotic ornithine decarboxylases. Another full ORF was found on the opposite strand downstream from the nspC gene. It encoded a protein of 69 amino acids with a calculated molecular mass of 7441 Da, which exhibited some weak similarity to ScrR, a repressor protein of V. alginolyticus, in the helix-turn-helix DNA binding domain, but did not appear to be expressed in the host cells.