Application of long amplicon propidium monoazide-PCR to assess the effects of temperature and background microbiota on pathogens in river water.
The decay rates of enteric waterborne pathogens were evaluated following the introduction of Yersinia enterocolitica, Salmonella enterica, Campylobacter jejuni and Arcobacter butzleri into river water at different temperatures (5, 15 and 25°C) for a period of 28 days. To improve the accuracy of the results a molecular viability assay, long amplicon propidium monoazide-polymerase chain reaction (PMA-PCR), was used to quantify the viable cell concentration and results from PCR with and without PMA were compared. As well, the effect of background microbiota was assessed for Y. enterocolitica and S. enterica by inoculating cells into sterile and non-sterile river water. Cell persistence was improved by up to 4 log for Y. enterocolitica and 4.5 log for S. enterica in sterile river water compared to natural river water, showing that the autochthonous biological activity in river water can accelerate the die-off of introduced bacteria. Results also showed that low temperature significantly improved the persistence of all four target bacteria in non-sterile river water. There was a more rapid decline in cell concentration in samples with PMA pretreatment; therefore using PMA-PCR analysis can provide more reliable data on viable/active enteric bacteria in aquatic microcosms and allows for improved assessment of pathogens in the environment.


Congenital disorders of glycosylation: a concise chart of glycocalyx dysfunction.
Glycosylation is a ubiquitous modification of lipids and proteins. Despite the essential contribution of glycoconjugates to the viability of all living organisms, diseases of glycosylation in humans have only been identified over the past few decades. The recent development of next-generation DNA sequencing techniques has accelerated the pace of discovery of novel glycosylation defects. The description of multiple mutations across glycosylation pathways not only revealed tremendous diversity in functional impairments, but also pointed to phenotypic similarities, emphasizing the interconnected flow of substrates underlying glycan assembly. The current list of 100 known glycosylation disorders provides an overview of the significance of glycosylation in human development and physiology.


Effects of signal transduction inhibitor 571 in acute myelogenous leukemia cells.
STI571 is a 2-phenylalaminopyrimidine derivative that inhibits c-abl, Bcr-Abl, and platelet-derived growth factor receptor tyrosine kinases. Recently, inhibition of stem cell factor (SCF)-induced c-kit phosphorylation and cell proliferation by STI571 was reported in the human myeloid cell line MO7e. Because approximately 70% of acute myelogenous leukemia (AML) cases are c-kit positive, we evaluated in vitro effects of STI571 on c-kit-positive cell lines and primary AML blast cells. At concentrations >5 microM, the drug marginally inhibited SCF-independent proliferation of cell lines and most of AML blasts. Treatment of AML cells with cytarabine and STI571 showed synergistic effect at low concentrations. Western blotting analysis documented a distinct band of M(r) 145,000 specific for c-kit in cell lines and in AML samples. There was no correlation between the level of the c-kit expression evaluated by Western blotting and percentage of c-kit-positive blasts as measured by flow cytometry. Neither in cell lines nor in primary AML cells, c-kit autophosphorylation was detectable under standard growth conditions. SCF-induced phosphorylation of c-kit in MO7e cells was inhibited by STI571. In a c-kit-positive AML-4 cell line, as well as in AML samples, c-kit phosphorylation was not induced by SCF exposure, suggesting that in these cases, the receptor could not be functionally activated. In conclusion, with the exception of MO7e, SCF did not induce phosphorylation of c-kit, and cell proliferation was not modulated in the presence of STI571. We did not detect any SCF-independent c-kit phosphorylation in our experimental systems. Consequently, STI571 exerted only a limited inhibitory effect on the cell growth.


Tilsotolimod: an investigational synthetic toll-like receptor 9 (TLR9) agonist for the treatment of refractory solid tumors and melanoma.
Cancer immunotherapy has seen tremendous strides in the past 15 years, with the introduction of several novel immunotherapeutic agents. Nevertheless, as clinical practice has shown, significant challenges remain with a considerable number of patients responding sub-optimally to available therapeutic options. Research has demonstrated the important immunoregulatory role of the tumor microenvironment (TME), with the potential to either hinder or promote an effective anti-tumor immune response. As such, scientific efforts have focused on investigating novel candidate immunomodulatory agents with the potential to alter the TME toward a more immunopotentiating composition. Herein, we discuss the novel investigational toll-like receptor 9 agonist tilsotolimod currently undergoing phase II and III clinical trials for advanced refractory cancer, highlighting its mode of action, efficacy, tolerability, and potential future applications in the treatment of cancer. To this effect, we conducted an exhaustive Web of Science and PubMed search to evaluate available research on tilsotolimod as of August 2021. With encouraging early clinical results demonstrating extensive TME immunomodulation and abscopal effects on distant tumor lesions, tilsotolimod has emerged as a potential candidate immunomodulatory agent with the possibility to augment currently available immunotherapy and provide novel avenues of treatment for patients with advanced refectory cancer.


Ocular hypotensive effect of oral palmitoyl-ethanolamide: a clinical trial.
To investigate the effect of oral palmitoyl-ethanolamide (PEA) on intraocular pressure (IOP) in primary open angle glaucoma (POAG) and ocular hypertension (OH). In a prospective, randomized, double-blind, crossover clinical trial, 42 patients with POAG or OH who were treated with timolol 0.5% and whose IOP was between 19 and 24 mm Hg received oral PEA (300-mg tablets twice a day) or placebo (PEA vehicle tablets twice a day) for 2 months (period 1), and, after a 2-month washout, received the other treatment for 1 month (period 2). IOP, best-corrected visual acuity, and visual field parameters were considered. After PEA treatment (mean baseline IOP, 21.6 ± 1.7 mm Hg), IOP was reduced by 3.2 ± 1.3 mm Hg at 1 month and by 3.5 ± 1.2 mm Hg (15.9% ± 5.1%) at 2 months (ANOVA, P < 0.001; both Tukey-Kramer, P < 0.01 vs. baseline); after placebo (mean baseline IOP, 21.5 ± 1.5 mm Hg), IOP was reduced by 0.4 ± 1.2 mm Hg at 1 month and by 0.3 ± 1.3 mm Hg at 2 months (t-test at both time points, P < 0.001 vs. PEA). No statistically significant vital signs, visual field, visual acuity changes, or adverse events were detected in either group. Systemic administration of PEA reduces IOP in patients with glaucoma and ocular hypertension. PEA could be a valuable tool for the treatment of glaucoma (http://www.umin.ac.jp/ctr/index/htm number, UMIN000002833).


Image-analysis based readout method for biochip: Automated quantification of immunomagnetic beads, micropads and patient leukemia cell.
For diagnosing and monitoring the progress of cancer, detection and quantification of tumor cells is utmost important. Beside standard bench top instruments, several biochip-based methods have been developed for this purpose. Our biochip design incorporates micron size immunomagnetic beads together with micropad arrays, thus requires automated detection and quantification of not only cells but also the micropads and the immunomagnetic beads. The main purpose of the biochip is to capture target cells having different antigens simultaneously. In this proposed study, a digital image processing-based method to quantify the leukemia cells, immunomagnetic beads and micropads was developed as a readout method for the biochip. Color, size-based object detection and object segmentation methods were implemented to detect structures in the images acquired from the biochip by a bright field optical microscope. It has been shown that manual counting and flow cytometry results are in good agreement with the developed automated counting. Average precision is 85 % and average error rate is 13 % for all images of patient samples, average precision is 99 % and average error rate is 1% for cell culture images. With the optimized micropad size, proposed method can reach up to 95 % precision rate for patient samples with an execution time of 90 s per image.


Recommendations on incurred sample stability (ISS) by GCC.
The topic of incurred sample stability (ISS) has generated considerable discussion within the bioanalytical community in recent years. The subject was an integral part of the seventh annual Workshop on Recent Issues in Bioanalysis (WRIB) held in Long Beach, CA, USA, in April 2013, and at the Global CRO Council for Bioanalysis (GCC) meeting preceding it. Discussion at both events focused on the use of incurred samples for ISS purposes in light of results from a recent GCC survey completed by member companies. This paper reports the consensus resulting from these discussions and serves as a useful reference for depicting ISS issues and concerns, summarizing the GCC survey results and providing helpful recommendations on ISS in the context of bioanalytical method development and application.


Phase 1 of the NIH Preprint Pilot: Testing the viability of making preprints discoverable in PubMed Central and PubMed.
The National Library of Medicine (NLM) launched a pilot in June 2020 to 1) explore the feasibility and utility of adding preprints to PubMed Central (PMC) and making them discoverable in PubMed and 2) to support accelerated discoverability of NIH-supported research without compromising user trust in NLM's widely used literature services. The first phase of the Pilot focused on archiving preprints reporting NIH-supported SARS-CoV-2 virus and COVID-19 research. To launch Phase 1, NLM identified eligible preprint servers and developed processes for identifying NIH-supported preprints within scope in these servers. Processes were also developed for the ingest and conversion of preprints in PMC and to send corresponding records to PubMed. User interfaces were modified for display of preprint records. NLM collected data on the preprints ingested and discovery of preprint records in PMC and PubMed and engaged users through focus groups and a survey to obtain direct feedback on the Pilot and perceptions of preprints. Between June 2020 and June 2022, NLM added more than 3,300 preprint records to PMC and PubMed, which were viewed 4 million times and 3 million times, respectively. Nearly a quarter of preprints in the Pilot were not associated with a peer-reviewed published journal article. User feedback revealed that the inclusion of preprints did not have a notable impact on trust in PMC or PubMed. NIH-supported preprints can be identified and added to PMC and PubMed without disrupting existing operations processes. Additionally, inclusion of preprints in PMC and PubMed accelerates discovery of NIH research without reducing trust in NLM literature services. Phase 1 of the Pilot provided a useful testbed for studying NIH investigator preprint posting practices, as well as knowledge gaps among user groups, during the COVID-19 public health emergency, an unusual time with heightened interest in immediate access to research results.


Anthropogenic environmental drivers of antimicrobial resistance in wildlife.
The isolation of antimicrobial resistant bacteria (ARB) from wildlife living adjacent to humans has led to the suggestion that such antimicrobial resistance (AMR) is anthropogenically driven by exposure to antimicrobials and ARB. However, ARB have also been detected in wildlife living in areas without interaction with humans. Here, we investigated patterns of resistance in Escherichia coli isolated from 408 wild bird and mammal faecal samples. AMR and multi-drug resistance (MDR) prevalence in wildlife samples differed significantly between a Sewage Treatment Plant (STP; wastes of antibiotic-treated humans) and a Farm site (antibiotic-treated livestock wastes) and Central site (no sources of wastes containing anthropogenic AMR or antimicrobials), but patterns of resistance also varied significantly over time and between mammals and birds. Over 30% of AMR isolates were resistant to colistin, a last-resort antibiotic, but resistance was not due to the mcr-1 gene. ESBL and AmpC activity were common in isolates from mammals. Wildlife were, therefore, harbouring resistance of clinical relevance. AMR E. coli, including MDR, were found in diverse wildlife species, and the patterns and prevalence of resistance were not consistently associated with site and therefore different exposure risks. We conclude that AMR in commensal bacteria of wildlife is not driven simply by anthropogenic factors, and, in practical terms, this may limit the utility of wildlife as sentinels of spatial variation in the transmission of environmental AMR.


Desulfonatronospira sulfatiphila sp. nov., and Desulfitispora elongata sp. nov., two novel haloalkaliphilic sulfidogenic bacteria from soda lakes.
Two novel haloalkaliphilic bacteria with dissimilatory sulfidogenic metabolism were recovered from syntrophic associations obtained from anaerobic sediments of hypersaline soda lakes in Kulunda Steppe (Altai, Russia). Strain ASO3-2T was a member of a sulfidogenic syntrophic association oxidizing acetate at extremely haloalkaline conditions, and was isolated in pure culture using formate as electron donor and sulfate as electron acceptor. It was identified as representing a novel member of the genus Desulfonatronospira within the Deltaproteobacteria. In contrast to the two known species of this genus, the novel isolate was able to grow with formate as electron donor and sulfate, as well as with sulfite, as electron acceptor. Strain Acr1T was a minor component in a soda lake syntrophic association converting benzoate to methane and acetate. It became dominant in a subculture fed with crotonate. While growing on crotonate, strain Acr1T formed unusually long cells filled with polyhydroxyalkanoate-like granules. Its metabolism was limited to fermentation of crotonate and pyruvate and the ability to utilize thiosulfate and sulfur/polysulfide as electron acceptor. Strain Acr1T was identified as representing a novel member of the genus Desulfitispora in the class Clostridia. Both isolates were obligately haloalkaliphilic with extreme salt tolerance. On the basis of phenotypic and phylogenetic analyses, the novel sulfidogenic isolates from soda lakes are proposed to represent two novel species: Desulfonatronospira sulfatiphila sp. nov. (ASO3-2T=DSM 100427=UNIQEM U993T) and Desulfitispora elongata sp. nov. (Acr1T=DSM 29990=UNIQEM U994T).


Cerebral hemodynamics in newborn infants exposed to speech sounds: a whole-head optical topography study.
Considerable knowledge on neural development related to speech perception has been obtained by functional imaging studies using near-infrared spectroscopy (optical topography). In particular, a pioneering study showed stronger left-dominant activation in the temporal lobe for (normal) forward speech (FW) than for (reversed) backward speech (BW) in neonates. However, it is unclear whether this stronger left-dominant activation for FW is equally observed for any language or is clearer for the mother tongue. We hypothesized that the maternal language elicits clearer activation than a foreign language in newborns because of their prenatal and/or few-day postnatal exposure to the maternal language. To test this hypothesis, we developed a whole-head optode cap for 72-channel optical topography and visualized the spatiotemporal hemodynamics in the brains of 17 Japanese newborns when they were exposed to FW and BW in their maternal language (Japanese) and in a foreign language (English). Statistical analysis showed that all sound stimuli together induced significant activation in the bilateral temporal regions and the frontal region. They also showed that the left temporal-parietal region was significantly more active for Japanese FW than Japanese BW or English FW, while no significant difference between FW and BW was shown for English. This supports our hypothesis and suggests that the few-day-old brain begins to become attuned to the maternal language. Together with a finding of equivalent activation for all sound stimuli in the adjacent measurement positions in the temporal region, these findings further clarify the functional organization of the neonatal brain.


Final results of a multi-institutional phase II trial of reirradiation with concurrent weekly cisplatin and cetuximab for recurrent or second primary squamous cell carcinoma of the head and neck.
The optimal regimen of chemotherapy and reirradiation (re-XRT) for recurrent head and neck squamous cell carcinoma (HNSCC) is controversial. We report the final outcomes of a multicenter phase II trial evaluating cetuximab and cisplatin-based chemotherapy concurrent with re-XRT for patients with recurrent HNSCC. Patients with unresectable recurrent disease or positive margins after salvage surgery arising within a previously irradiated field with KPS ≥ 70 were eligible for this trial. Cetuximab 400 mg/m2 was delivered as a loading dose in week 1 followed by weekly cetuximab 250 mg/m2 and cisplatin 30 mg/m2 concurrent with 6 weeks of intensity-modulated radiotherapy to a dose of 60-66 Gy in 30 daily fractions. Patients who previously received both concurrent cetuximab and cisplatin with radiation or who received radiotherapy less than 6 months prior were ineligible. From 2009 to 2013, 48 patients enrolled on this trial, 2 did not receive any protocol treatment. Of the remaining 46 patients, 34 were male and 12 female, with a median age of 62 years (range 36-85). Treatment was feasible and only 1 patient did not complete the treatment course. Common grade 3 or higher acute toxicities were lymphopenia (46%), pain (22%), dysphagia (13%), radiation dermatitis (13%), mucositis (11%) and anorexia (11%). There were no grade 5 acute toxicities. Eight grade 3 late toxicities were observed, four of which were swallowing related. With a median follow-up of 1.38 years, the 1-year overall survival (OS) was 60.4% and 1-year recurrence-free survival was 34.1%. On univariate analysis, OS was significantly improved with young age (P = 0.01). OS was not associated with radiation dose, surgery before re-XRT or interval from prior XRT. Concurrent cisplatin and cetuximab with re-XRT is feasible and offers good treatment outcomes for patients with high-risk features. Younger patients had significantly improved OS. NCT00833261.


Physics-based forecasting of man-made earthquake hazards in Oklahoma and Kansas.
Reinjection of saltwater, co-produced with oil, triggered thousands of widely felt and several damaging earthquakes in Oklahoma and Kansas. The future seismic hazard remains uncertain. Here, we present a new methodology to forecast the probability of damaging induced earthquakes in space and time. In our hybrid physical-statistical model, seismicity is driven by the rate of injection-induced pressure increases at any given location and spatial variations in the number and stress state of preexisting basement faults affected by the pressure increase. If current injection practices continue, earthquake hazards are expected to decrease slowly. Approximately 190, 130 and 100 widely felt M ≥ 3 earthquakes are anticipated in 2018, 2019 and 2020, respectively, with corresponding probabilities of potentially damaging M ≥ 5 earthquakes of 32, 24 and 19%. We identify areas where produced-water injection is more likely to cause seismicity. Our methodology can be used to evaluate future injection scenarios intended to mitigate seismic hazards.


Prevalence of antibodies against Mycobacterium avium subsp paratuberculosis among beef cow-calf herds.
To estimate the prevalence of Mycobacterium avium subsp paratuberculosis infection among cows on beef operations in the United States. Cross-sectional seroprevalence study. Sample Population-A convenience sample of 380 herds in 21 states. Serum samples were obtained from 10,371 cows and tested for antibodies to M avium subsp paratuberculosis with a commercial ELISA. Producers were interviewed to collect data on herd management practices. 30 (7.9%) herds had 1 or more animals for which results of the ELISA were positive; 40 (0.4%) of the individual cow samples yielded positive results. None of the herd management practices studied were found to be associated with whether any animals in the herd would be positive for antibodies to M avium subsp paratuberculosis. Results suggest that the prevalence of antibodies to M avium subsp paratuberculosis among beef cows in the United States is low. Herds with seropositive animals were widely distributed geographically.


Association of the IRF5 risk haplotype with high serum interferon-alpha activity in systemic lupus erythematosus patients.
A haplotype of the interferon regulatory factor 5 (IRF5) gene has been associated with the risk of developing systemic lupus erythematosus (SLE), and our previous studies have demonstrated that high levels of serum interferon-alpha (IFNalpha) activity are a heritable risk factor for SLE. The aim of this study was to determine whether the IRF5 SLE risk haplotype mediates the risk of SLE by predisposing patients to the development of high levels of serum IFNalpha activity. IFNalpha levels in 199 SLE patients of European and Hispanic ancestry were measured with a sensitive functional reporter cell assay. The rs2004640, rs3807306, rs10488631, and rs2280714 single-nucleotide polymorphisms (SNPs) in IRF5 were genotyped in these patients. Haplotypes were categorized as SLE risk, neutral, or protective based on published data. SLE patients with risk/risk and risk/neutral IRF5 genotypes had higher serum IFNalpha activity than did those with protective/protective and neutral/protective genotypes (P = 0.025). This differential effect of IRF5 genotype on serum IFNalpha levels was driven largely by SLE patients who were positive for either anti-RNA binding protein (anti-RBP) or anti-double-stranded DNA (anti-dsDNA) autoantibodies (P = 0.012 for risk/risk or risk/neutral versus protective/protective or neutral/protective). The rs3807306 genotype was independently associated with high serum IFNalpha in this autoantibody group. We found no difference in IFNalpha activity according to IRF5 genotype in patients lacking either type of autoantibody or in patients positive for both classes of autoantibody. The IRF5 SLE risk haplotype is associated with higher serum IFNalpha activity in SLE patients, and this effect is most prominent in patients positive for either anti-RBP or anti-dsDNA autoantibodies. This study demonstrates the biologic relevance of the SLE risk haplotype of IRF5 at the protein level.


Acute and sublethal effects of sequential exposure to the pesticide azinphos-methyl on juvenile earthworms (Eisenia andrei).
The use of organophosphate pesticides is an integral part of commercial farming activities and these substances have been implicated as a major source of environmental contamination and may impact on a range of non-target fauna. The extent to which soil dwelling non-target organisms are affected by exposure to the organophosphate azinphos-methyl was investigated through monitoring selected biomarker responses and life cycle effects under laboratory conditions in the earthworm Eisenia andrei. Standard acute toxicity tests were conducted followed by a sequential exposure regime experiment, in order to assess the effects of multiple pesticide applications on biomarker (cholinesterase activity and neutral red retention time), life-cycle (growth and reproduction) and behaviour (avoidance and burrowing activity) responses. The present study indicates that the time between exposure events was a more important variable than concentration and that a longer interval between exposures may mitigate the effects of pesticide exposure provided that the exposure concentration is low. Additionally, it was shown that E. andrei was unable to avoid the presence of azinphos-methyl in soil, even at concentrations as high as 50% of the LC(50) value, indicating that the presence of azinphos-methyl in the soil pose a realistic threat to earthworms and other soil dwelling organisms. The ChE inhibition test showed a high percentage inhibition of the enzyme in all exposure groups that survived and NRR times of exposed organisms were lower than that of the controls. The present study yielded important results that contribute to the understanding of biological impacts of pesticide pollution on the environment. Extrapolating these results can aid in optimising pesticide application regimes to mitigate the environmental effects thereof and thus ensuring sustained soil biodiversity in agricultural areas.


Scanning electron microscope study of bacteria associated with the rumen epithelium of sheep.
Examination of the rumen epithelium of sheep by scanning electron microscopy revealed bacteria associated with the epithelial surface. Comparison of epithelial surfaces from 10 sheep revealed areas that were consistently densely covered with bacteria and other areas where the cover was consistently light. The bacterial populations were frequently of mixed morphological types, but areas populated with a single type were also observed. This finding, together with the discovery of bacterial forms not previously described in rumen contents, suggests that a specific flora may exist on the rumen epithelial surface. The functional significance of such a population is discussed.


Precise targeting of cancer metastasis using multi-ligand nanoparticles incorporating four different ligands.
Metastasis displays a highly heterogeneous cellular population with cancer cells continuously evolving. As a result, a single-ligand nanoparticle cannot account for the continuously changing expression of targetable biomarkers over time and space. To effectively direct nanoparticles to metastasis, we developed a multi-ligand nanoparticle by using four different types of ligands on the same nanoparticle that target biomarkers on the endothelium associated with metastatic disease. These vascular targets included αvβ3 integrin, P-selectin, EGFR and fibronectin. Using terminal and in vivo imaging studies, the targeting performance of the multi-ligand nanoparticles was compared to the single-ligand nanoparticle variants. All four single-ligand nanoparticle variants achieved significant targeting of lung metastasis in the 4T1 mouse model of breast cancer metastasis with about 2.5% of the injected dose being deposited into metastasis. A dual-ligand nanoparticle resulted in a nearly 2-fold higher deposition into lung metastases than its single-ligand counterparts. The multi-ligand nanoparticle significantly outperformed its targeting nanoparticle counterparts achieving a deposition of ∼7% of its injected nanoparticles into lung metastases. Using the high sensitivity of radionuclide imaging, PET imaging showed that a multi-ligand nanoparticle labeled with [18F]fluoride was able to precisely target metastatic disease at its very early stage of development in three different animal models of metastatic breast cancer.


An interstitial deletion-insertion involving chromosomes 2p25.3 and Xq27.1, near SOX3, causes X-linked recessive hypoparathyroidism.
X-linked recessive hypoparathyroidism, due to parathyroid agenesis, has been mapped to a 906-kb region on Xq27 that contains 3 genes (ATP11C, U7snRNA, and SOX3), and analyses have not revealed mutations. We therefore characterized this region by combined analysis of single nucleotide polymorphisms and sequence-tagged sites. This identified a 23- to 25-kb deletion, which did not contain genes. However, DNA fiber-FISH and pulsed-field gel electrophoresis revealed an approximately 340-kb insertion that replaced the deleted fragment. Use of flow-sorted X chromosome-specific libraries and DNA sequence analyses revealed that the telomeric and centromeric breakpoints on X were, respectively, approximately 67 kb downstream of SOX3 and within a repetitive sequence. Use of a monochromosomal somatic cell hybrid panel and metaphase-FISH mapping demonstrated that the insertion originated from 2p25 and contained a segment of the SNTG2 gene that lacked an open reading frame. However, the deletion-insertion [del(X)(q27.1) inv ins (X;2)(q27.1;p25.3)], which represents a novel abnormality causing hypoparathyroidism, could result in a position effect on SOX3 expression. Indeed, SOX3 expression was demonstrated, by in situ hybridization, in the developing parathyroid tissue of mouse embryos between 10.5 and 15.5 days post coitum. Thus, our results indicate a likely new role for SOX3 in the embryonic development of the parathyroid glands.


Folate/Vitamin B12 Supplementation Combats Oxidative Stress-Associated Carcinogenesis in a Rat Model of Colon Cancer.
Folate and vitamin B12 deficiency is associated with depletion of the major intracellular antioxidant glutathione, and oxidative stress is emerging as an etiological mechanism for colon cancer. Azoxymethane (AOM), a potent carcinogen, induces colon cancer in rats by causing pathophysiological changes and oxidative stress. We investigated the synergistic effect of folate and vitamin B12 supplementation against AOM-induced carcinogenesis and oxidative stress in rat colon. Adult male rats were distributed into four groups: 1) Basal diet only; 2) AOM injection (15 mg/kg once per week in weeks 5 and 6); 3) Folate and vitamin B12 supplemented diet; 4) Folate and B12 diet with AOM injection. After 16 weeks, rats were sacrificed, colon tissue dissected, indicators of oxidative stress were measured, and immunohistochemical and ultrastructural changes were evaluated. AOM-injected rats showed oxidative stress, evident by glutathione depletion, oxidation of cellular proteins, and DNA oxidative damage. AOM increased mucosal levels of antiapoptotic and proapoptotic proteins Bcl2 and Bax and caused ultrastructure changes in colonic cell organelles. Folate and vitamin B12 supplementation decreased the level of oxidative stress and ameliorated the cytotoxic effects of AOM. In this in vivo experimental model of colon cancer, folate and vitamin B12 supplementation combats carcinogen-induced oxidative stress.


The coral reef crisis: the critical importance of<350 ppm CO2.
Temperature-induced mass coral bleaching causing mortality on a wide geographic scale started when atmospheric CO(2) levels exceeded approximately 320 ppm. When CO(2) levels reached approximately 340 ppm, sporadic but highly destructive mass bleaching occurred in most reefs world-wide, often associated with El Niño events. Recovery was dependent on the vulnerability of individual reef areas and on the reef's previous history and resilience. At today's level of approximately 387 ppm, allowing a lag-time of 10 years for sea temperatures to respond, most reefs world-wide are committed to an irreversible decline. Mass bleaching will in future become annual, departing from the 4 to 7 years return-time of El Niño events. Bleaching will be exacerbated by the effects of degraded water-quality and increased severe weather events. In addition, the progressive onset of ocean acidification will cause reduction of coral growth and retardation of the growth of high magnesium calcite-secreting coralline algae. If CO(2) levels are allowed to reach 450 ppm (due to occur by 2030-2040 at the current rates), reefs will be in rapid and terminal decline world-wide from multiple synergies arising from mass bleaching, ocean acidification, and other environmental impacts. Damage to shallow reef communities will become extensive with consequent reduction of biodiversity followed by extinctions. Reefs will cease to be large-scale nursery grounds for fish and will cease to have most of their current value to humanity. There will be knock-on effects to ecosystems associated with reefs, and to other pelagic and benthic ecosystems. Should CO(2) levels reach 600 ppm reefs will be eroding geological structures with populations of surviving biota restricted to refuges. Domino effects will follow, affecting many other marine ecosystems. This is likely to have been the path of great mass extinctions of the past, adding to the case that anthropogenic CO(2) emissions could trigger the Earth's sixth mass extinction.


Calmodulin activates neuronal nitric oxide synthase by enabling transitions between conformational states.
We recently showed that inducible nitric oxide synthase conformational intermediates can be resolved via FMN fluorescence lifetimes. Here we show that neuronal NOS activation by calmodulin removes constraints favoring a closed 'input state', increasing occupation of other states and facilitating conformational transitions. The 90 ps FMN input state lifetime distinguishes it from ∼4 ns 'open' states in which FMN does not interact strongly with other groups, or 0.9 ns output states in which FMN interacts with ferriheme. Enablement of the conformational cycle is an important paradigm for control in nNOS and related enzymes, and may extend to other control modalities.


Secretory carcinoma of the breast: a genetically defined carcinoma entity.
Secretory carcinomas (SBC) are characterized by their characteristic histomorphology and more favorable prognosis compared to invasive ductal carcinoma of usual type (IDC). On this basis, 13 SBCs are evaluated by molecular and immunohistochemical (IH) methods. 13 SBCs and 4 IDCs were analyzed for ETV6-NTRK3 gene fusion by reverse transcriptase-polymerase chain reaction (RT-PCR) and by Fluorescence in situ Hybridization (FISH). 8 of 13 microdissected SBCs with evaluable DNA were evaluated for genetic alterations (GA) by comparative genomic hybridization (CGH). IH included estrogen-receptor (ER), progesterone-receptor (PR), Her-2/neu and Ki-67 (MIB-1) in all 13 cases. Molecular and immunohistochemical results in SBCs were compared with previous data regarding immunohistochemical and molecular characteristics of IDCs. 12 of 13 (92 %) SBC cases, but not IDCs expressed the ETV6-NTRK3 fusion gene which encodes a chimeric tyrosine kinase. Retroviral transfer of ETV6-NTRK3 (EN) into murine mammary epithelial cells resulted in transformed cells that readily formed epithelial tumors in nude mice. CGH revealed an average of 2.0 GAs (range 0-6), including recurrent gains of chromosome 8q and 1q and losses of 22q. Four SBCs were positive for ER and 2 were positive for PR. The mean MIB-1-labeling index was 11.4% (range: <1-34%). Her-2/ neu protein overexpression was detected in 1 case (score 3+). Compared to previous findings in IDCs, SBCs are characterized by the recurrent expression of ETV6-NTRK3 fusion gene, a relatively low number of GAs, low proliferative rate, infrequent Her-2/ neu protein overexpression and a lower rate of steroid hormone receptor expression. These results support the hypothesis that SBCs have immunohistochemical and genetic features that specifically distinguish them from IDCs.


Combination of PI3K/mTOR inhibitors: antitumor activity and molecular correlates.
The phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR pathway is a major target for cancer therapy. As a strategy to induce the maximal inhibition of this pathway in cancer cells, we combined allosteric mTOR inhibitors (rapamycin and RAD001) with a dual PI3K/mTOR kinase inhibitor (PI-103). Both in vitro and in vivo, the combination exhibited more activity than single agents in human ovarian and prostate cancer cells that harbor alterations in the pathway. At the molecular level, combined inhibition of mTOR prevented the rebound activation of Akt that is seen after treatment with rapamycin and its analogues and caused more sustained inhibition of Akt phosphorylation. Furthermore, the combination strongly inhibited the expression of PI3K/Akt/mTOR downstream proteins. In particular, it showed greater activity than the single agents in inhibiting the phosphorylation of 4EBP1, both in vitro and in vivo, resulting in selective inhibition of CAP-dependent translation. A proteomic approach was used to confirm the identification of c-Myc as the key regulator for the reduction in downstream proteins affected by the combined inhibition of mTOR. In conclusion, the combination of a catalytic and an allosteric inhibitor of mTOR shows greater activity, without a concomitant increase in toxicity, than either drug alone, and this may have therapeutic implications for inhibiting this pathway in the clinical setting.


Diagnostic accuracy of case-finding questions to identify perinatal depression.
Guidelines for perinatal mental health care recommend the use of two case-finding questions about depressed feelings and loss of interest in activities, despite the absence of validation studies in this context. We examined the diagnostic accuracy of these questions and of a third question about the need for help asked of women receiving perinatal care. We evaluated self-reported responses to two case-finding questions against an interviewer-assessed diagnostic standard (DSM-IV criteria for major depressive disorder) among 152 women receiving antenatal care at 26-28 weeks' gestation and postnatal care at 5-13 weeks after delivery. Among women who answered "yes" to either question, we assessed the usefulness of asking a third question about the need for help. We calculated sensitivity, specificity and likelihood ratios for the two case-finding questions and for the added question about the need for help. Antenatally, the two case-finding questions had a sensitivity of 100% (95% confidence interval [CI] 77%-100%), a specificity of 68% (95% CI 58%-76%), a positive likelihood ratio of 3.03 (95% CI 2.28-4.02) and a negative likelihood ratio of 0.041 (95% CI 0.003-0.63) in identifying perinatal depression. Postnatal results were similar. Among the women who screened positive antenatally, the additional question about the need for help had a sensitivity of 58% (95% CI 38%-76%), a specificity of 91% (95% CI 78%-97%), a positive likelihood ratio of 6.86 (95% CI 2.16-21.7) and a negative likelihood ratio of 0.45 (95% CI 0.25-0.80), with lower sensitivity and higher specificity postnatally. Negative responses to both of the case-finding questions showed acceptable accuracy for ruling out perinatal depression. For positive responses, the use of a third question about the need for help improved specificity and the ability to rule in depression.


Mindfulness increases recall of self-threatening information.
Previous research has suggested that we tend to show impaired memory for self-threatening information, an effect known as mnemic neglect. Mnemic neglect is believed to be due to shallow processing or inhibition of self-threatening information. Mnemic neglect, however, could also be an example of experiential avoidance and mindfulness training has been demonstrated to counteract experiential avoidance. The current study was designed to negate experiential avoidance on a memory task via mindfulness training and attempt to increase recall of self-threatening information. Participants were exposed to a short intervention, either mindfulness or unfocused attention, before being instructed to read and later recall self-referent behaviors. The findings indicated that recall of self-threatening and other self-referent information was increased following the mindfulness but not unfocused attention intervention. The utility of mindfulness as a strategy for negating the experiential avoidance normally associated with self-threatening information and increasing memory performance are discussed.


DNA polymerase 3'→5' exonuclease activity: Different roles of the beta hairpin structure in family-B DNA polymerases.
Proofreading by the bacteriophage T4 and RB69 DNA polymerases requires a β hairpin structure that resides in the exonuclease domain. Genetic, biochemical and structural studies demonstrate that the phage β hairpin acts as a wedge to separate the primer-end from the template strand in exonuclease complexes. Single amino acid substitutions in the tip of the hairpin or deletion of the hairpin prevent proofreading and create "mutator" DNA polymerases. There is little known, however, about the function of similar hairpin structures in other family B DNA polymerases. We present mutational analysis of the yeast (Saccharomyces cerevisiae) DNA polymerase δ hairpin. Deletion of the DNA polymerase δ hairpin (hpΔ) did not significantly reduce DNA replication fidelity; thus, the β hairpin structure in yeast DNA polymerase δ is not essential for proofreading. However, replication efficiency was reduced as indicated by a slow growth phenotype. In contrast, the G447D amino acid substitution in the tip of the hairpin increased frameshift mutations and sensitivity to hydroxyurea (HU). A chimeric yeast DNA polymerase δ was constructed in which the T4 DNA polymerase hairpin (T4hp) replaced the yeast DNA polymerase δ hairpin; a strong increase in frameshift mutations was observed and the mutant strain was sensitive to HU and to the pyrophosphate analog, phosphonoacetic acid (PAA). But all phenotypes - slow growth, HU-sensitivity, PAA-sensitivity, and reduced fidelity, were observed only in the absence of mismatch repair (MMR), which implicates a role for MMR in mediating DNA polymerase δ replication problems. In comparison, another family B DNA polymerase, DNA polymerase ɛ, has only an atrophied hairpin with no apparent function. Thus, while family B DNA polymerases share conserved motifs and general structural features, the β hairpin has evolved to meet specific needs.


Imaging Findings of an Epidermoid Cyst with Malignant Transformation to Squamous Cell Carcinoma.
We report imaging findings of a 64-year-old male patient with a ruptured epidermoid cyst (EC) known to be constant over the 23-year follow-up and showing malignant transformation to squamous cell carcinoma (SCC). Computed tomography (CT) and magnetic resonance imaging (MRI) findings including diffusion weighted imaging (DWI), 1H+MR spectroscopy (MRS), dynamic susceptibility contrast perfusion (DSC) MRI of EC, and its rare complications are presented together with a review of the literature. Fluid-lowattenuated- inversion-recovery (FLAIR) and T1-weighted images with gadolinium are the best sequences together with DWI to show the relationship of the EC, the SCC and the border between. Primary brain SCC enhances mostly ring-like or peripherally, but diffuse enhancement is also possible. To our knowledge, no MRS and DSC findings have been reported in the literature yet.


Dating violence and self-efficacy for delayed sex among adolescents in Cape Town, South Africa.
In South Africa, dating violence is known to be widespread among adolescents, and is therefore a major public health issue because of its association with sexual risk behaviours. The objective of the study was to examine the relationship between dating violence and self-efficacy for delayed sex among school-going adolescents in Cape Town, South Africa. The study is based on analyses of data from a school-based health education programme targeting sexual and reproductive health issues.The study involved 3,655 school-going adolescents aged between 12 and 17 in Cape Town, South Africa. The data was collected by means of a self-administered questionnaire composed of 153 items on sexual and reproductive health, dating violence as well as sociodemographic characteristics. The results indicated that males showed a higher percentage of both dating violence victimization and perpetration, as compared to females. It was also found that adolescents from lower socio-economic backgrounds were more likely to be the victims of dating violence as compared to those from a higher socio-economic background. Female learners showed higher levels of self-efficacy for delayed sex than their male counterparts. Although the result revealed that there was a significant association between self-efficacy for delayed sex and socio-economic status, this link decreased with age. It is concluded that educational programmes aimed solely at improving self-efficacy for delayed sex is insufficient. Such programmes must also aim at preventing dating violence and equipping adolescents with the skills to negotiate their way out of dating violence.


Biomimetic corneal stroma using electro-compacted collagen.
Engineering substantia propria (or stroma of cornea) that mimics the function and anatomy of natural tissue is vital for in vitro modelling and in vivo regeneration. There are, however, few examples of bioengineered biomimetic corneal stroma. Here we describe the construction of an orthogonally oriented 3D corneal stroma model (3D-CSM) using pure electro-compacted collagen (EC). EC films comprise aligned collagen fibrils and support primary human corneal stromal cells (hCSCs). Cell-laden constructs are analogous to the anatomical structure of native human cornea. The hCSCs are guided by the topographical cues provided by the aligned collagen fibrils of the EC films. Importantly, the 3D-CSM are biodegradable, highly transparent, glucose-permeable and comprise quiescent hCSCs. Gene expression analysis indicated the presence of aligned collagen fibrils is strongly coupled to downregulation of active fibroblast/myofibroblast markers α-SMA and Thy-1, with a concomitant upregulation of the dormant keratocyte marker ALDH3. The 3D-CSM represents the first example of an optimally robust biomimetic engineered corneal stroma that is constructed from pure electro-compacted collagen for cell and tissue support. The 3D-CSM is a significant advance for synthetic corneal stroma engineering, with the potential to be used for full-thickness and functional cornea replacement, as well as informing in vivo tissue regeneration. STATEMENT OF SIGNIFICANCE: This manuscript represents the first example of a robust, transparent, glucose permeable and pure collagen-based biomimetic 3D corneal stromal model (3D-CSM) constructed from pure electro-compacted collagen. The collagen fibrils of 3D-CSM are aligned and orthogonally arranged, mimicking native human corneal stroma. The alignment of collagen fibrils correlates with the direction of current applied for electro-compaction and influences human corneal stromal cell (hCSC) orientation. Moreover, 3D-CSM constructs support a corneal keratocyte phenotype; an essential requirement for modelling healthy corneal stroma. As-prepared 3D-CSM hold great promise as corneal stromal substitutes for research and translation, with the potential to be used for full-thickness cornea replacement.


Phenomenology of intentional repetitive behaviors in obsessive-compulsive disorder and Tourette's disorder.
Recent evidence suggests that obsessive-compulsive disorder (OCD) and Tourette's disorder are related and have overlapping clinical features. The purpose of this study was to test the following hypotheses regarding intentional repetitive behaviors in these two disorders: (1) In OCD without comorbid Tourette's, they are preceded by cognitive phenomena and autonomic anxiety, but not sensory phenomena, and (2) in Tourette's without comorbid OCD, they are preceded by sensory phenomena, but not cognitive phenomena nor autonomic anxiety. Fifteen adult OCD outpatients without tics and 17 adult Tourette's outpatients without OCD were evaluated with a structured interview. Questions assessed cognitive, sensory, and affective experiences related to intentional repetitive behaviors. Five of 17 Tourette's subjects were excluded because they had only unintentional or occasionally intentional tics. All OCD patients reported some cognitions preceding their intentional repetitive behaviors, whereas only 2 of 12 Tourette's patients reported cognitions. In comparison, all Tourette's patients reported sensory phenomena preceding repetitive behaviors, and none of the OCD patients reported such sensations. In addition, 13 OCD patients reported at least mild autonomic anxiety associated with their repetitive behaviors, whereas no Tourette's patients reported such symptoms. Intentional repetitive behaviors in OCD differ from those in Tourette's and are associated with cognitive and autonomic phenomena. Sensory phenomena preceded intentional repetitive behaviors in Tourette's but not in OCD patients. The dimensions examined in this study (cognition, sensory phenomena, and autonomic anxiety) may represent valid clinical factors for characterization of repetitive behaviors in OCD and Tourette's.


CALGB 30704 (Alliance): A randomized phase II study to assess the efficacy of pemetrexed or sunitinib or pemetrexed plus sunitinib in the second-line treatment of advanced non-small-cell lung cancer.
Second-line chemotherapy for advanced non-small cell lung cancer (NSCLC) improves survival modestly but new strategies are needed. This trial was designed to evaluate an antivascular endothelial growth factor strategy with or without standard chemotherapy in previously treated NSCLC. Patients with stage IIIB/IV NSCLC with performance status 0 to 1 progressive after first-line chemotherapy were eligible for randomization to pemetrexed, sunitinib, or the combination. Patients were stratified by performance status, stage, and sex. Primary objective was 18-week progression-free survival (PFS) rate; secondary objectives included response, overall survival (OS), and toxicity. Target accrual was 225. The study was terminated early because of decreasing accrual rates. Between April 2008 and September 2011, 130 patients were registered and randomized; of this, 125 patients were treated. Baseline characteristics in the three arms were well balanced. Toxicity was higher in the sunitinib-containing arms. The 18-week PFS rate in the pemetrexed, sunitinib, and combination arms was 54% (95% confidence interval [CI], 40-71), 37% (95% CI, 25-54), and 48% (95% CI, 35-66), respectively (p = 0.25). Median PFS in the pemetrexed, sunitinib, and combination arms in months was 4.9 (2.1-8.8), 3.3 (2.3-4.2), and 3.7 (2.5-5.8), respectively (p = 0.18). There was an overall statistically significant difference in OS between the three arms: median OS in months was 10.5 (8.3-20.2) for pemetrexed, 8.0 (6.8-13.5) for sunitinib, and 6.7 (4.1-10.1) for the combination (p = 0.03). Pemetrexed had a superior toxicity profile to either sunitinib or the combination of pemetrexed and sunitinib. The 18-week PFS rate was not significantly different between the arms. OS was significantly better with pemetrexed alone compared with the two sunitinib-containing arms, with the doublet performing worst for OS.


Therapeutic potential of dichloroacetate for pyruvate dehydrogenase complex deficiency.
We reviewed the use of oral dichloroacetate (DCA) in the treatment of children with congenital lactic acidosis caused by mutations in the pyruvate dehydrogenase complex (PDC). The case histories of 46 subjects were analyzed with regard to diagnosis, clinical presentation and response to DCA. DCA decreased blood and cerebrospinal fluid lactate concentrations, and was generally well tolerated. DCA may be particularly effective in children with PDC deficiency by stimulating residual enzyme activity and, consequently, cellular energy metabolism. A controlled trial is needed to determine the definitive role of DCA in the management of this devastating disease.


Sucrose availability on the aerial part of the plant promotes morphogenesis and flowering of Arabidopsis in the dark.
Conditions to promote dark morphogenesis and flower-ing in Arabidopsis have previously been limited to liquid cultures and to a few laboratory ecotypes. We have obtained development and flowering of Arabidopsis plants under complete darkness by growing them on vertical Petri dishes containing solid agar medium with sucrose. Under these conditions, all the ecotypes tested were able to develop, giving rise to etiolated plants that flowered after producing a certain number of leaves. Dark-grown plants showed similarities with phytochrome-deficient mutants and were different from de-etiolated or constitutive photomorphogenesis mutants such as det and cop. Late- and early-flowering ecotypes, showing large differences in flowering time and leaf number under long days, flowered with a similar number of leaves when grown in the dark. Rapid dark flowering of late-flowering ecotypes was not an effect of darkness but the result of the interaction between dark and sucrose availability at the aerial part of the plant, since sucrose also had an effect when plants were grown in the light. Gibberellin-deficient and insensitive mutants were delayed in the initiation of flowers in the dark, indicating a role for these hormones in flowering promotion in the dark. The late-flowering phenotype of mutants at different loci of the autonomous and long-day-dependent flowering induction pathways was rescued in dark growth conditions. However, the late-flowering phenotype of ft and fwa mutants was not rescued by sucrose either in the dark or in the light, suggesting a different role for these genes in flowering induction.


Update in handheld electronic resources for evidence-based practice in the community setting.
To provide an update on the handheld electronic resources for evidence-based practice (EBP) in the community setting. Electronic resources for EBP in the community setting were identified by compiling the commonly used, well-established resources and by searching MEDLINE and other Internet sites. Search terms included evidence-based medicine, evidence-based practice, resources, and abstraction. Only sources available for personal digital assistants were included. Three databases were identified that provided abstraction and evaluation of the medical literature for the handheld platform. Content, features, ease of use, system requirements, and costs of each resource were evaluated. FIRSTConsult, InfoRetriever, and UpToDate were evaluated, and the utility of each in the community pharmacy setting was evaluated by tracking a clinically relevant example through each system. FIRSTConsult provides evidence-based information organized by diagnosis but is not searchable on the handheld platform. InfoRetriever focuses on searchable evidence-based summaries, while UpToDate includes comprehensive topic reviews. The latter 2 platforms have large system memory requirements. All 3 sources provide evidence-based abstraction of the medical literature for the PDA platform, convenient for use at the point of care in community pharmacy. While users may select a particular resource based on unique features, each provides evidence-based abstraction of the medical literature that is a practical approach to EBP in the community pharmacy setting.


Cannabidiol as a Treatment for Chronic Pain: A Survey of Patients' Perspectives and Attitudes.
Cannabis products have become easily available and accessible after decriminalization of cannabis for recreational and medicinal use in many states. Cannabidiol (CBD) has been of increasing interest to patients and is being used to self-medicate a variety of ailments. However, very limited information is available to patients and providers to form an educated opinion regarding its indicated use to treat the many conditions this substance has been implied to be helpful for. The aim of this survey was to learn about participants' attitudes and views towards cannabis-based medicine (CBM) with a focus on perception of "CBD" and its potential role for pain management. We recruited survey participants from seven pain management clinics in Southern California to learn about their knowledge, beliefs, and personal experience with CBD products. After Institutional Review Board (IRB) review, an internet survey platform was utilized to administer the survey online. A total of 253 participants answered the survey. Participants were 45.4 ± 13.8 (Mean ± SD) years of age, the majority identified as white (56.1%), had an annual household income of less than $20,000, and were primarily insured by Medicare (22.5%) or Medicaid (43.9%). Among participants, 62.0% reported trying a CBD product [including products containing delta-9-tetrahydrocannabinol (THC)]. The majority responded that these products have helped their pain (59.0%) and allowed them to reduce their pain medications (67.6%), including opioids (53.7%). They reported believing that CBD was a good treatment option (71.1%), not harmful (74.9%), and not addictive (65.3%). About half of participants (51.9%) report that they would be more comfortable with their physician prescribing CBD products. The overall attitude and experience of participants regarding CBD is reported as positive, while 91.9% of people expressed a desire to learn more about it. In summary, most participants expressed a positive attitude about CBD products as a treatment option, reported positive outcomes when used for multiple different conditions, and would prefer to obtain information about and prescription for CBD from their physicians.


Elevated plasma levels of human adrenomedullin in cardiovascular, respiratory, hepatic and renal disorders.
1. Adrenomedullin is a potent vasodilating peptide first isolated from phaeochromocytoma and adrenal medulla but also found in the heart, lungs and kidneys. It may also be a paracrine factor because endothelial and smooth muscle cells synthesize adrenomedullin as well as express the receptors. Adrenomedullin induces vasorelaxation by activating adenylate cyclase and also by stimulating the release of nitric oxide. 2. We have developed a specific radioimmunoassay and measured the immunoreactivity of human adrenomedullin in the plasma of 58 male subjects: eight with essential hypertension, 12 with heart failure, 10 with ascites due to cirrhosis, 12 with chronic renal failure, four with hypoxia due to chronic obstructive pulmonary disease and 12 control subjects. 3. Plasma levels (mean +/- SEM) in patients with essential hypertension (16.3 +/- 1.9 pmol/l), congestive heart failure (17.5 +/- 2.8 pmol/l) and renal failure (17.7 +/- 2.5 pmol/l) were raised compared with control subjects (7.8 +/- 1.4 pmol/l, P < 0.05), confirming previous reports. 4. In addition, we observed that plasma levels of adrenomedullin were significantly raised in patients with ascites due to liver cirrhosis (15.5 +/- 1.9 pmol/l) and chronic obstructive pulmonary disease with hypoxia (20.0 +/- 1.5 pmol/l). 5. We concluded that the plasma level of adrenomedullin is raised in a variety of diseases.


Laparoscopic resection of gastric diverticulum.
Gastric diverticulum is a rare disorder. The laparoscopic approach may be ideal for the resection of gastric diverticulum in some cases. The authors believe this to be the first reported case of gastric diverticulum resected laparoscopically. A 59-year-old woman was admitted with indigestion and epigastric pain. Upper gastrointestinal series showed a single diverticulum (3 x 2 cm) on the posterior wall of the upper part of the stomach. The neck of the diverticulum was relatively narrow. Gastroduodenoscopy revealed that food residues were impacted within the diverticular pouch. Other areas of the stomach and the duodenum were normal. Abdominal ultrasonography showed no other pathologic conditions in the upper part of abdomen. On operation, the lesser sac was entered by division of the greater omentum along the avascular plane of the transverse colonic attachment. The location of the diverticulum was confirmed by intraoperative gastroscopy. Using a 5-cannula techniques, the diverticulum was resected with an endoscopic linear stapler device. Pathologic examination of the resected specimen demonstrated chronic superficial gastritis. Flatus was passed out on the first postoperative day, diet was started from the second day, and the patient was discharged on the fifth day without problems.


Inflammation in induced sputum after aluminium oxide exposure: an experimental chamber study.
Workers in aluminium production are exposed to a complex mixture of particles and gases potentially harmful to the airways, among them aluminium oxide (Al2O3). With the use of an exposure chamber, we aimed to examine the effects of short-term controlled exposure to Al2O3 on lung function and inflammatory markers in healthy volunteers. 15 men (age 19-31) were exposed in random order to clean air or Al2O3 particles (3.8-4.0 mg/m(3)) for 2 h including 30 min exercise (stationary bike, 75 W). The permissible exposure level (PEL) for Al2O3 by Occupational Safety and Health Administration, USA, is 5 mg/m(3) time weighted average (TWA). Sham and particle exposures were separated by at least 2 weeks. Spirometry was carried out, and induced sputum and blood samples were collected 48 h before and 4 and 24 h after exposure. Levels of sputum neutrophils (mean (±SEM)) was increased 24 h post-Al2O3 vs pre-Al2O3 exposure (43% (4) vs 31% (4), p=0.01) and the protein level of interleukin (IL)-8 had a 4.8 (0.9)-fold change increase 24 h after exposure (p<0.01). Following Al2O3 exposure, gene signatures in sputum were significantly increased related to several pathways. The present study suggests that controlled exposure to Al2O3 particles at levels below PEL (TWA) induces airway inflammation in healthy humans marked by elevated neutrophils and elevated IL-8. In addition, increased expression of genes associated with several biological processes was observed in sputum. Interestingly, inhaled Al2O3-induced effects were localised to the airways and not systemic.


Regulatory effect of caffeic acid phenethyl ester on type I collagen and interferon-gamma in bleomycin-induced pulmonary fibrosis in rat.
Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease of unknown etiology. Recent investigations have demonstrated that the impaired immune response is a common characteristic feature of IPF. Unfortunately, no definitive and effective drug treatment is available that could improve or at least inhibit the progressive course of this fatal disease. That is why one of the main priorities of pulmonary fibrosis investigations is to identify novel and effective molecular targets for preventive and therapeutic interventions. caffeic acid phenethyl ester (CAPE) is one of the most interesting bioactive compounds extracted from bee propolis. It has been shown that CAPE has an antioxidant activity and modulatory impact on immune system. Accordingly, the aim of the present study was to investigate the regulatory effects of CAPE on the levels of type I collagen (COL-1) and Interferon-gamma (IFN-γ) in bleomycin (BLM)-induced pulmonary fibrosis. Immunohistochemistry procedure was employed to assess the effects of CAPE on lung tissue. In this study, male Sprague-Dawley rats were divided into 5 groups (n=8) included 1: Positive control group: bleomycin (BLM). 2: Negative (saline) control group. 3, 4: Treatment groups of 1 and 2: BLM+CAPE (5 and 10 μmol/kg/day, respectively). (5: Sham group: CAPE (10 μmol/kg/day). BLM application resulted in significant changes in the level of studied parameters as compared to the controls. CAPE could decrease type I collagen concentration, modulate IFN-γ level, increase the animals' body weight and decrease the lung index dose-dependently, compared with model group. In conclusion, CAPE may provide a novel therapeutic target for treating pulmonary fibrosis.


The Endocannabinoid System as a Target in Cancer Diseases: Are We There Yet?
The endocannabinoid system (ECS) has been placed in the anti-cancer spotlight in the last decade. The immense data load published on its dual role in both tumorigenesis and inhibition of tumor growth and metastatic spread has transformed the cannabinoid receptors CB1 (CB1R) and CB2 (CB2R), and other members of the endocannabinoid-like system, into attractive new targets for the treatment of various cancer subtypes. Although the clinical use of cannabinoids has been extensively documented in the palliative setting, clinical trials on their application as anti-cancer drugs are still ongoing. As drug repurposing is significantly faster and more economical than de novo introduction of a new drug into the clinic, there is hope that the existing pharmacokinetic and safety data on the ECS ligands will contribute to their successful translation into oncological healthcare. CB1R and CB2R are members of a large family of membrane proteins called G protein-coupled receptors (GPCR). GPCRs can form homodimers, heterodimers and higher order oligomers with other GPCRs or non-GPCRs. Currently, several CB1R and CB2R-containing heteromers have been reported and, in cancer cells, CB2R form heteromers with the G protein-coupled chemokine receptor CXCR4, the G protein-coupled receptor 55 (GPR55) and the tyrosine kinase receptor (TKR) human V-Erb-B2 Avian Erythroblastic Leukemia Viral Oncogene Homolog 2 (HER2). These protein complexes possess unique pharmacological and signaling properties, and their modulation might affect the antitumoral activity of the ECS. This review will explore the potential of the endocannabinoid network in the anti-cancer setting as well as the clinical and ethical pitfalls behind it, and will develop on the value of cannabinoid receptor heteromers as potential new targets for anti-cancer therapies and as prognostic biomarkers.


Structural basis of pathogen recognition by an integrated HMA domain in a plant NLR immune receptor.
Plants have evolved intracellular immune receptors to detect pathogen proteins known as effectors. How these immune receptors detect effectors remains poorly understood. Here we describe the structural basis for direct recognition of AVR-Pik, an effector from the rice blast pathogen, by the rice intracellular NLR immune receptor Pik. AVR-PikD binds a dimer of the Pikp-1 HMA integrated domain with nanomolar affinity. The crystal structure of the Pikp-HMA/AVR-PikD complex enabled design of mutations to alter protein interaction in yeast and in vitro, and perturb effector-mediated response both in a rice cultivar containing Pikp and upon expression of AVR-PikD and Pikp in the model plant Nicotiana benthamiana. These data reveal the molecular details of a recognition event, mediated by a novel integrated domain in an NLR, which initiates a plant immune response and resistance to rice blast disease. Such studies underpin novel opportunities for engineering disease resistance to plant pathogens in staple food crops.


Sensory adaptation of leukocytes to chemotactic peptides.
The morphology and behavior of polymorphonuclear leukocytes (PMNs) were studied after rapid changes in the concentration of a chemotactic factor N-formylnorleucylleucylphenylalanine (f-NorleuLeuPhe) (Schiffmann et al., 1975, Proc. Natl. Acad. Sci. U. S. A. 72:1059--1062). After an increase in peptide concentration, the cells round, form lamellipodia or ruffles over most of their surface, and stop locomotion. These changes are transient. After a delay, the cells, still in the presence of peptide, withdraw most of the ruffles and resume locomotion, forming ruffles only at their front. Cells repeat the transient generalized ruffling upon further increase in peptide concentration. The behavioral changes occur over the same dose range as binding to a saturable receptor. The duration of the transient response after a concentration increase is roughly proportional to the increase in the number of cell receptors occupied as a result of the concentration change. Decreasing the concentration of peptide causes the cells to round transiently and form blebs before they recommence locomotion. The transient nature of these aspects of the cell's responsiveness to chemotactic factors appears to be due to adaptation by the cells. The ability to adapt to the concentration of a chemotactic factor may be important in leukocyte chemotaxis.


Comparative Transcriptomic Analysis of Two Actinorhizal Plants and the Legume Medicago truncatula Supports the Homology of Root Nodule Symbioses and Is Congruent With a Two-Step Process of Evolution in the Nitrogen-Fixing Clade of Angiosperms.
Root nodule symbiosis (RNS) is a symbiotic interaction established between angiosperm hosts and nitrogen-fixing soil bacteria in specialized organs called root nodules. The host plants provide photosynthate and the microsymbionts supply fixed nitrogen. The origin of RNS represents a major evolutionary event in the angiosperms, and understanding the genetic underpinnings of this event is of major economic and agricultural importance. Plants that engage in RNS are restricted to a single angiosperm clade known as the nitrogen-fixing clade (NFC), yet occur in multiple lineages scattered within the NFC. It has been postulated that RNS evolved in two steps: a gain-of-predisposition event occurring at the base of the NFC, followed by a gain-of-function event in each host plant lineage. Here, we first explore the premise that RNS has evolved from a single common background, and then we explore whether a two-step process better explains the evolutionary origin of RNS than either a single-step process, or multiple origins. We assembled the transcriptomes of root and nodule of two actinorhizal plants, Ceanothus thyrsiflorus and Datisca glomerata. Together with the corresponding published transcriptomes of the model legume Medicago truncatula, the gene expression patterns in roots and nodules were compared across the three lineages. We found that orthologs of many genes essential for RNS in the model legumes are expressed in all three lineages, and that the overall nodule gene expression patterns were more similar to each other than expected by random chance, a finding that supports a common evolutionary background for RNS shared by the three lineages. Moreover, phylogenetic analyses suggested that a substantial portion of the genes experiencing selection pressure changes at the base of the NFC also experienced additional changes at the base of each host plant lineage. Our results (1) support the occurrence of an event that led to RNS at the base of the NFC, and (2) suggest a subsequent change in each lineage, most consistent with a two-step origin of RNS. Among several conserved functions identified, strigolactone-related genes were down-regulated in nodules of all three species, suggesting a shared function similar to that shown for arbuscular mycorrhizal symbioses.


Genetic analysis of H2, the structural gene for phase-2 flagellin in Salmonella.
Non-flagellate H2 mutants were isolated from a phase-2 stable strain, SJW806 H1-gt- H2-enxon vh2-, a derivative of Salmonella typhimurium. By transductional crosses a deletion map and a recombination map of the H2 gene were made. There are three regions especially rich in nonflagellate mutational sites. By the use of the deletion map, mutational sites of 21 flagellar shape mutants were also determined. Most of them were located at two regions which coincide with two of the three regions rich in non-flagellate mutational sites. A gene, vh2, is closely linked to the promoter side of the H2 gene. Three-factor transductional crosses showed that the vh2 gene was on the left of the H2 gene in the present map. The H2 gene forms part of an operon with the distal gene rh1 which specifies the H1 repressor. Thus, a polarity effect of the H2 mutations on the expression of the rh1 gene was examined by observing whether a wild-type H1 allele introduced into the H2 mutants was expressed or not. Many of the H2 mutations were polar, and most of the strongly polar mutations were located in the left (promoter-proximal) half of the H2 gene, while most of the mutations in the right half of the gene were weakly polar or non-polar.


Smurf2 participates in human trophoblast cell invasion by inhibiting TGF-beta type I receptor.
Successful embryo implantation depends on the ability of the trophoblast cells to invade the endometrium and the receptivity of the endometrium. Unlike tumor invasion, trophoblast invasion is spatio-temporaly restricted. Transforming growth factor (TGF)-beta is a key inhibitory factor in the invasion of early trophoblast cells. Smad ubiquitination regulatory factor 2 (Smurf2), a HECT type E3 ubiquitin ligase, is an important regulator of the TGF-beta signaling pathway, targeting TGF-beta receptors and various Smads for proteasome-mediated degradation. In this context, we wished to determine whether Smurf2 has a physiological role during embryo implantation, especially in trophoblast invasion. We examined the spatio-temporal expression of Smurf2 in human placental villi and the function of Smurf2 in trophoblast cell migration and invasion in a model system involving a human extravillous trophoblast cell line, HTR-8/SVneo. Results from RT-PCR and immunohistochemical studies showed that expression of Smurf2 in placental villi was the highest during the first trimester and decreased as the pregnancy progressed. Overexpression of Smurf2 in HTR-8/SVneo cells reduced TGF-beta type I receptor levels, and enhanced cell migration and invasion. Conversely, RNA interference-mediated downregulation of Smurf2 resulted in a significant increase in TGF-beta type I receptor protein levels. However, the levels of Smad2, another potential target of Smurf2, remained unchanged. In conclusion, the present study suggests that Smurf2 promotes trophoblast cell migration and invasion, and this function may involve downregulation of TGF-beta type I receptor.


Revisiting a theory of cerebellar cortex.
Long-term depression at parallel fiber-Purkinje cell synapses plays a principal role in learning in the cerebellum, which acts as a supervised learning machine. Recent experiments demonstrate various forms of synaptic plasticity at different sites within the cerebellum. In this article, we take into consideration synaptic plasticity at parallel fiber-molecular layer interneuron synapses as well as at parallel fiber-Purkinje cell synapses, and propose that the cerebellar cortex performs reinforcement learning, another form of learning that is more capable than supervised learning. We posit that through the use of reinforcement learning, the need for explicit teacher signals for learning in the cerebellum is eliminated; instead, learning can occur via responses from evaluative feedback. We demonstrate the learning capacity of cerebellar reinforcement learning using simple computer simulations of delay eyeblink conditioning and the cart-pole balancing task.


The Recent Trend in Diagnosis and Treatment of Chronic Low Back Pain.
Development of evidence-based medicine has made a big change in diagnosis and treatment of chronic low back pain. The recent trend is assessed through a review of literature. The articles published in these 10 years are reviewed, and important points are examined. In diagnosis, challenges for history taking and limit of imaging or clinical guidelines are revealed. In treatment, cognitive behavioral treatment and exercises are proved effective. Sleep disturbance has recently attracted attention as a factor associated with low back pain. Cost-effectiveness of diagnosis and treatment modalities has come to be emphasized. Diagnosis and treatment of chronic low back pain have been significantly changing. Multidisciplinary and multidimensional approach is essential. Chronic low back pain should be treated as a total pain, not a local pain.


Effect of the NK(3) receptor antagonist, talnetant, on rectal sensory function and compliance in healthy humans.
Visceral hypersensitivity is important in the pathophysiology of irritable bowel syndrome and thus a target for modulation in drug development. Neurokinin (NK) receptors, including NK(3) receptors, are expressed in the motor and sensory systems of the digestive tract. The aim of this study was to compare the effects of two different doses (25 and 100 mg) of the NK(3) receptor antagonist, talnetant (SB223412) with placebo on rectal sensory function and compliance in healthy volunteers studied at two centres. Rectal barostat tests were performed on 102 healthy volunteers, randomized to receive either oral talnetant 25 or 100 mg or placebo over 14-17 days. Studies were performed on three occasions: day 1 immediately prior to 1st dose, day 1 4 h postdose, and after 14- to17-day therapy. Compliance, and pressure thresholds for first sensation, urgency, discomfort and pain were measured using ascending method of limits, and sensory intensity ratings for gas, urgency, discomfort and pain determined during four random phasic distensions (12, 24, 36 and 48 mmHg). Talnetant had no effect on rectal compliance, sensory thresholds or intensity ratings compared with placebo. In general, the results obtained at the two centres differed minimally, with intensity scores at one centre consistently somewhat lower. At the doses tested, talnetant has no effect on rectal compliance or distension-induced rectal sensation in healthy participants.


The impacts of the early outset of the COVID-19 pandemic on climate change research: Implications for policy-making.
Since January 2020, the COVID-19 pandemic has dominated the media and exercises pressure on governments worldwide. Apart from its effects on economies, education systems and societies, the pandemic has also influenced climate change research. This paper examines the extent to which COVID-19 has influenced climate change research worldwide during the first wave at the beginning of 2020 and how it is perceived to exploit it in the future. This study utilised an international survey involving those dedicated to climate change science and management research from Academia, Government, NGOs, and international agencies in 83 countries. The analysis of responses encompasses four independent variables: Institutions, Regions, Scientific Areas, and the level of economic development represented by the Human Development Index (HDI). Results show that: (1) COVID-19 modified the way the surveyed researchers work, (2) there are indicators that COVID-19 has already influenced the direction of climate change and adaptation policy implementation, and (3) respondents perceived (explicitly concerning the COVID-19 lockdowns of March-April 2020), that the pandemic has drawn attention away from climate policy. COVID- 19 has influenced the agenda of climate change research for more than half of the respondents and is likely to continue in the future, suggesting that the impacts on their research will still be felt for many years. The paper concludes by outlining critical implications for policy-making.


Long-term acceptability, durability and bio-efficacy of ZeroVector® durable lining for vector control in Papua New Guinea.
This study examined the acceptability, durability and bio-efficacy of pyrethroid-impregnated durable lining (DL) over a three-year period post-installation in residential homes across Papua New Guinea (PNG). ZeroVector® ITPS had previously been installed in 40 homes across four study sites representing a cross section of malaria transmission risk and housing style. Structured questionnaires, DL visual inspections and group interviews (GIs) were completed with household heads at 12- and 36-months post-installation. Three DL samples were collected from all households in which it remained 36-months post-installation to evaluate the bio-efficacy of DL on Anopheles mosquitoes. Bio-efficacy testing followed WHO guidelines for the evaluation of indoor residual spraying. The DL was still intact in 86 and 39% of study homes at the two time periods, respectively. In homes in which the DL was still intact, 92% of household heads considered the appearance at 12-months post installation to be the same as, or better than, that at installation compared to 59% at 36-months post-installation. GIs at both time points confirmed continuing high acceptance of DL, based in large part of the perceived attractiveness and functionality of the material. However, participants frequently asserted that they, or their family members, had ceased or reduced their use of mosquito nets as a result of the DL installation. A total of 16 houses were sampled for bio-efficacy testing across the 4 study sites at 36-months post-installation. Overall, combining all sites and samples, both knock-down at 30 min and mortality at 24 h were 100%. The ZeroVector® DL installation remained highly acceptable at 36-months post-installation, the material and fixtures proved durable and the efficacy against malaria vectors did not decrease. However, the DL material had been removed from over 50% of the original study homes 3 years post-installation, largely due to deteriorating housing infrastructure. Furthermore, the presence of the DL installation appeared to reduce ITN use among many participating householders. The study findings suggest DL may not be an appropriate vector control method for large-scale use in the contemporary PNG malaria control programme.


Extended phase-matching properties of periodically poled potassium niobate crystals for mid-infrared polarization-entangled photon-pair generation.
We report the extended phase-matching (EPM) properties of two kinds of periodically poled potassium niobate (KNbO3 or KN) crystals (i.e., periodic 180°- and 90°-domain structures) that are highly useful for the generation of polarization-entangled photon pairs in the mid-infrared (IR) spectral region. Under the degenerate Type II spontaneous parametric downconversion process satisfying the EPM condition, an input single photon with a frequency of 2ω generates a pair of synchronized photons with identical frequencies of ω that are orthogonally polarized with respect to each other (i.e., the frequency-coincident, polarization-entangled biphoton states). Our simulation results illustrate that the EPM is achievable in the mid-IR spectral region: at the wavelengths of 3.80 μm and 4.03 μm for periodic 90°- and 180°-domain structures, respectively. We will describe in detail the EPM properties of both cases in terms of interaction types and the corresponding nonlinear optic coefficients, phase-matching bandwidths, and domain poling periods. The calculated EPM bandwidths are much broader than 200 nm in the mid-IR for both cases, exhibiting a great potential for nonlinear-optic signal processing in quantum communication systems operating in the mid-IR bands.


Functional consequences of mutations in the autophagy genes in the pathogenesis of Crohn's disease.
Genome-wide association studies have highlighted a number of genes involved in autophagy, which are of potential importance in the pathogenesis of Crohn's disease (CD). The associated polymorphisms in ATG16L1 and IRGM have been confirmed, and functional studies have begun to shed light on how they link to CD pathogenesis. In this review we consider the most salient aspects of this rapidly expanding field.


Dietary pectin-derived acidic oligosaccharides improve the pulmonary bacterial clearance of Pseudomonas aeruginosa lung infection in mice by modulating intestinal microbiota and immunity.
A predominantly T-helper type 2 (Th2) immune response is critical in the prognosis of pulmonary Pseudomonas aeruginosa infection. But the mucosal and systemic immune responses can be influenced by the intestinal microbiota. We assessed the effect of microbiota compositional changes induced by a diet enriched in 5% acidic oligosaccharides derived from pectin (pAOS) on the immune response and outcome of chronic pulmonary P. aeruginosa infection in mice. pAOS promoted Th1 polarization by increasing interferon γ release, upregulating t-bet gene expression, decreasing interleukin 4 secretion, and downregulating gata3 gene expression. pAOS also sustained the release of keratinocyte chemoattractant, recruited polynuclear leukocytes and macrophages, stimulated M1 macrophage activation and interleukin 10 release, and decreased tumor necrosis factor α release in the lung. These effects led to increased bacterial clearance after the first and second P. aeruginosa infections. pAOS modified the intestinal microbiota by stimulating the growth of species involved in immunity development, such as Bifidobacterium species, Sutturella wadsworthia, and Clostridium cluster XIVa organisms, and at the same time increased the production of butyrate and propionate. These results suggest that pAOS may have beneficial effects by limiting the number and severity of pulmonary exacerbations in patients chronically infected with P. aeruginosa, such as individuals with cystic fibrosis.


Potential of Salivary Biomarkers in Autism Research: A Systematic Review.
The diagnostic process for autism spectrum disorders (ASD) is based on a behavioral analysis of the suspected individual. Despite intensive research, no specific and valid biomarker has been identified for ASD, but saliva, with its advantages such as non-invasive collection, could serve as a suitable alternative to other body fluids. As a source of nucleic acid of both human and microbial origin, protein and non-protein molecules, saliva offers a complex view on the current state of the organism. Additionally, the use of salivary markers seems to be less complicated not only for ASD screening but also for revealing the etiopathogenesis of ASD, since enrolling neurotypical counterparts willing to participate in studies may be more feasible. The aim of the presented review is to provide an overview of the current research performed on saliva in relation to ASD, mutual complementing, and discrepancies that result in difficulties applying the observed markers in clinical practice. We emphasize the methodological limitations of saliva collection and processing as well as the lack of information regarding ASD diagnosis, which is critically discussed.


Endometrial effects of tibolone.
The Tibolone Histology of the Endometrium and Breast Endpoints Study is a multicenter, randomized, double-blind study designed to address the conflicting reports in the literature about the endometrial safety of tibolone (1.25 or 2.5 mg/d). Tibolone was compared with continuous combined conjugated equine estrogen (CEE) plus medroxyprogesterone acetate (MPA) (0.625 + 2.5 mg/d). Subjects were randomized in a 1:1:2 ratio to tibolone 1.25 mg/d, 2.5 mg/d, and CEE/MPA, respectively. The one-sided 95% confidence interval (CI) has been evaluated for the incidence of abnormal endometrial histology (hyperplasia or carcinoma) and hyperplasia and carcinoma separately for each of the two treatment groups and the treatment groups combined after 1 and 2 yr of treatment with tibolone, compared with CEE/MPA. A total of 3240 women were randomized, with 3224 receiving at least one dose of study medication. The incidence and upper one-sided 95% CI for the incidence of abnormal endometrium (hyperplasia or carcinoma), and hyperplasia and carcinoma separately, were calculated at end point, yr 1, and yr 2. The incidence (upper one-sided 95% CI) of abnormal endometrium at end point was 0.0 (0.5), 0.0 (0.4), and 0.2 (0.5) in the tibolone 1.25 mg, 2.5 mg, and CEE/MPA groups, respectively. During the entire treatment period, amenorrhea was reported more frequently with tibolone 1.25 mg (78.7%) and 2.5 mg (71.4%) than CEE/MPA (44.9%). The Tibolone Histology of the Endometrium and Breast Endpoints Study results confirm previous findings that tibolone does not induce endometrial hyperplasia or carcinoma in postmenopausal women, and it is associated with a better vaginal bleeding profile than CEE/MPA.


Deficits in adults with autism spectrum disorders when processing multiple objects in dynamic scenes.
People with autism spectrum disorders (ASD) process visual information in a manner that is distinct from typically developing individuals. They may be less sensitive to people's goals and, more generally, focus on visual details instead of the entire scene. To examine these differences, people with and without ASD were asked to detect changes in dynamic scenes with multiple elements. Participants viewed a brief video of a person or an inanimate object (the "figure") moving from one object to another; after a delay, they reported whether a second video was the same or different. Possible changes included the figure, the object the figure was moving from, or the object the figure was moving toward (the "goal"). We hypothesized that individuals with ASD would be less sensitive to changes in scenes with people, particularly elements that might be the person's goal. Alternately, people with ASD might attend to fewer elements regardless of whether the scene included a person. Our results indicate that, like controls, people with ASD noticed a change in the "goal" object at the end of a person's movement more often than the object at the start. However, the group with ASD did not undergo the developmental improvement that was evident typically when detecting changes in both the start and end objects. This atypical development led to deficits in adults with ASD that were not specific to scenes with people or to "goals." Improvements in visual processing that underlie mature representation of scenes may not occur in ASD, suggesting that late developing brain processes are affected.


The Asp(272)-Glu(282) region of platelet glycoprotein Ibalpha interacts with the heparin-binding site of alpha-thrombin and protects the enzyme from the heparin-catalyzed inhibition by antithrombin III.
Platelet glycoprotein Ib (GpIb) mediates interaction with both von Willebrand factor and thrombin. Thrombin binds to GpIb via its heparin-binding site (HBS) (De Candia, E., De Cristofaro, R., De Marco, L., Mazzucato, M., Picozzi, M., and Landolfi, R. (1997) Thromb. Haemostasis 77, 735-740; De Cristofaro, R., De Candia, E., Croce, G., Morosetti, R., and Landolfi, R. (1998) Biochem. J. 332, 643-650). To identify the thrombin-binding domain on GpIbalpha, we examined the effect of GpIbalpha(1-282), a GpIbalpha fragment released by the cobra venom mocarhagin on the heparin-catalyzed rate of thrombin inhibition by antithrombin III (AT). GpIbalpha(1-282) inhibited the reaction in a dose-dependent and competitive fashion. In contrast, the GpIbalpha(1-271) fragment, produced by exposing GpIbalpha(1-282) to carboxypeptidase Y, had no effect on thrombin inhibition by the heparin-AT complex. Measurements of the apparent equilibrium constant of the GpIbalpha(1-282) binding to thrombin as a function of different salts (NaCl and tetramethyl-ammonium chloride) concentration (0.1-0.2 M) indicated a large salt dependence (Gamma(+/-) = -4.5), similar to that pertaining to the heparin binding to thrombin. The importance of thrombin HBS in its interaction with GpIbalpha was confirmed using DNA aptamers, which specifically bind to either HBS (HD22) or the fibrinogen recognition site of thrombin (HD1). HD22, but not HD1, inhibited thrombin binding to GpIbalpha(1-282). Furthermore, the proteolytic derivative gamma(T)-thrombin, which lacks the fibrinogen recognition site, binds to GpIbalpha via its intact HBS in a reaction that is inhibited by HD22. Neither alpha- nor gamma(T)-thrombin bound to GpIbalpha(1-271), suggesting that the Asp(272)-Glu(282) region of GpIbalpha may act as a "heparin-like" ligand for the thrombin HBS, thereby inhibiting heparin binding to thrombin. It was also demonstrated that intact platelets may dose-dependently inhibit the heparin-catalyzed thrombin inhibition by AT at enzyme concentrations <5 nM. Altogether, these findings show that thrombin HBS binds to the region of GpIbalpha involving the Asp(272)-Glu(282) segment, protecting the enzyme from the inactivation by the heparin-AT system.


Org 214007-0: a novel non-steroidal selective glucocorticoid receptor modulator with full anti-inflammatory properties and improved therapeutic index.
Glucocorticoids (GCs) such as prednisolone are potent immunosuppressive drugs but suffer from severe adverse effects, including the induction of insulin resistance. Therefore, development of so-called Selective Glucocorticoid Receptor Modulators (SGRM) is highly desirable. Here we describe a non-steroidal Glucocorticoid Receptor (GR)-selective compound (Org 214007-0) with a binding affinity to GR similar to that of prednisolone. Structural modelling of the GR-Org 214007-0 binding site shows disturbance of the loop between helix 11 and helix 12 of GR, confirmed by partial recruitment of the TIF2-3 peptide. Using various cell lines and primary human cells, we show here that Org 214007-0 acts as a partial GC agonist, since it repressed inflammatory genes and was less effective in induction of metabolic genes. More importantly, in vivo studies in mice indicated that Org 214007-0 retained full efficacy in acute inflammation models as well as in a chronic collagen-induced arthritis (CIA) model. Gene expression profiling of muscle tissue derived from arthritic mice showed a partial activity of Org 214007-0 at an equi-efficacious dosage of prednisolone, with an increased ratio in repression versus induction of genes. Finally, in mice Org 214007-0 did not induce elevated fasting glucose nor the shift in glucose/glycogen balance in the liver seen with an equi-efficacious dose of prednisolone. All together, our data demonstrate that Org 214007-0 is a novel SGRMs with an improved therapeutic index compared to prednisolone. This class of SGRMs can contribute to effective anti-inflammatory therapy with a lower risk for metabolic side effects.


Rational design of catalysts for asymmetric diamination reaction using transition state modeling.
The stereoselective synthesis of 1,2-diamines has remained a formidable challenge. A recent palladium-catalyzed asymmetric diamination of conjugated double bonds using di-tert-butyldiaziridinone appears promising. The axially chiral binol phosphoramidite ligands are successful in offering high enantioselectivity. The density functional theory investigations revealed that the energies of the stereocontrolling transition states for the C-N bond formation depend on a number of weak non-covalent interactions such as C-H···π, C-H···O and anagostic interactions. We envisaged that the modulation in these interactions in the transition states, through subtle changes in chiral phosphoramidite substituents, could be exploited toward steering the stereoselectivity. The effect of systematic modifications on both 3,3' positions of the binol as well as on the amido nitrogen on the stereochemical outcome is predicted. It is identified that high enantioselectivity requires a balance between the nature of the substituents on binol and amido groups. The reduced size of the amido substituents demands increased bulk on the binol whereas lowering the size on the binol demands increased bulk on the amido for higher stereoselectivity. The substituent at the α-position of the amido group is found to be vital and appears to be a hot spot for modifications. These insights derived from studies on the stereocontrolling transition states could help improve the catalytic efficacies in palladium-catalyzed asymmetric diamination reactions.


Impact of formal training on agreement of videofluoroscopic swallowing study interpretation across and within disciplines.
Formal agreement studies on interpretation of the videofluoroscopic swallowing study (VFSS) procedure among speech-language pathologists, radiology house officers, and staff radiologists have not been pursued. Each of these professions participates in the procedure, interprets the examination, and writes separate reports on the findings. The aim of this study was to determine reliability of interpretation between and within the disciplines and to determine if structured training improved reliability. Thirteen speech-language pathologists (SLPs), ten diagnostic radiologists (RADs) and twenty-one diagnostic radiology house officers (HOs) participated in this study. Each group viewed 24 VFSS samples and rated the presence or absence of seven aberrant swallowing features as well as the presence of dysphagia and identification of oral dysphagia, pharyngeal dysphagia, or both. During part two, the groups were provided with a training session on normal and abnormal swallowing, using different VFSS samples from those in part one, followed by re-rating of the original 24 VFSS samples. A generalized estimating equations (GEE) approach with a binomial link function was used to examine each question separately. For each cluster of tests, as example, all pairwise comparisons between the three groups in the pretraining period, a Hochberg's correction for multiple testing was used to determine significance. A GEE approach with a binomial link function was used to compare the premeasure to postmeasure for each of the three groups of raters stratified by experience. The primary result revealed that the HO group scored significantly lower than the SLP and RAD group on identification of the presence of dysphagia (p = 0.008; p = 0.001, respectively), identification of oral phase dysphagia (p = 0.003; p = 0.001, respectively), and identification of both oral and pharyngeal phase dysphagia, (p = 0.014, p = 0.001, respectively) pretraining. Post training there was no statistically significant difference between the three groups on identification of dysphagia and identification of combined oral and pharyngeal dysphagia. Formal training to identify oropharyngeal dysphagia characteristics appears to improve accuracy of interpretation of the VFSS procedure for radiology house officers. Consideration to include formal training in this area for radiology residency training programs is recommended.


Constituents of the essential oil of Suregada zanzibariensis leaves are repellent to the mosquito, Anopheles gambiae s.s.
In traditional African communities, repellent volatiles from certain plants generated by direct burning or by thermal expulsion have played an important role in protecting households against vectors of malaria and other diseases. Previous research on volatile constituents of plants has shown that some are good sources of potent mosquito repellents. In this bioprospecting initiative, the essential oil of leaves of the tree, Suregada zanzibariensis Verdc. (Angiospermae: Euphobiaceae) was tested against the mosquito, Anopheles gambiae s.s. Giles (Diptera: Culicidae) and found to be repellent. Gas chromatography (GC), GC-linked mass spectrometry (GC-MS) and, where possible, GC-co-injections with authentic compounds, led to the identification of about 34 compounds in the essential oil. About 56% of the constituents were terpenoid ketones, mostly methyl ketones. Phenylacetaldehyde (14.4%), artemisia ketone (10.1%), (1S)-(-)-verbenone (12.1%) and geranyl acetone (9.4%) were the main constituents. Apart from phenylacetaldehyde, repellent activities of the other main constituents were higher than that of the essential oil. The blends of the main constituents in proportions found in the essential oil were more repellent to An. gambiae s.s. than was the parent oil (p < 0.05), and the presence of artemisia ketone in the blend caused a significant increase in the repellency of the resulting blend. These results suggested that blends of some terpenoid ketones can serve as effective An. gambiae s.s. mosquito repellents.