The structural and mechanistic bases for the viral resistance to allosteric HIV-1 integrase inhibitor pirmitegravir.

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are investigational antiretroviral agents that potently impair virion maturation by inducing hyper-multimerization of IN and inhibiting its interaction with viral genomic RNA. The pyrrolopyridine-based ALLINI pirmitegravir (PIR) has recently advanced into phase 2a clinical trials. Previous cell culture-based viral breakthrough assays identified the HIV-1(Y99H/A128T IN) variant that confers substantial resistance to this inhibitor. Here, we have elucidated the unexpected mechanism of viral resistance to PIR. Although both Tyr99 and Ala128 are positioned within the inhibitor binding V-shaped cavity at the IN catalytic core domain (CCD) dimer interface, the Y99H/A128T IN mutations did not substantially affect the direct binding of PIR to the CCD dimer or functional oligomerization of full-length IN. Instead, the drug-resistant mutations introduced a steric hindrance at the inhibitor-mediated interface between CCD and C-terminal domain (CTD) and compromised CTD binding to the CCDY99H/A128T + PIR complex. Consequently, full-length INY99H/A128T was substantially less susceptible to the PIR-induced hyper-multimerization than the WT protein, and HIV-1(Y99H/A128T IN) conferred >150-fold resistance to the inhibitor compared with the WT virus. By rationally modifying PIR, we have developed its analog EKC110, which readily induced hyper-multimerization of INY99H/A128T in vitro and was ~14-fold more potent against HIV-1(Y99H/A128T IN) than the parent inhibitor. These findings suggest a path for developing improved PIR chemotypes with a higher barrier to resistance for their potential clinical use.IMPORTANCEAntiretroviral therapies save the lives of millions of people living with HIV (PLWH). However, the evolution of multi-drug-resistant viral phenotypes is a major clinical problem, and there are limited or no treatment options for heavily treatment-experienced PLWH. Allosteric HIV-1 integrase inhibitors (ALLINIs) are a novel class of antiretroviral compounds that work by a unique mechanism of binding to the non-catalytic site on the viral protein and inducing aberrant integrase multimerization. Accordingly, ALLINIs potently inhibit both wild-type HIV-1 and all drug-resistant viral phenotypes that have so far emerged against currently used therapies. Pirmitegravir, a highly potent and safe investigational ALLINI, is currently advancing through clinical trials. Here, we have elucidated the structural and mechanistic bases behind the emergence of HIV-1 integrase mutations in infected cells that confer resistance to pirmitegravir. In turn, our findings allowed us to rationally develop an improved ALLINI with substantially enhanced potency against the pirmitegravir-resistant virus.

A Discussion of Building a Smart SHM Platform for Long-Span Bridge Monitoring.

This paper explores the development of a smart Structural Health Monitoring (SHM) platform tailored for long-span bridge monitoring, using the Forth Road Bridge (FRB) as a case study. It discusses the selection of smart sensors available for real-time monitoring, the formulation of an effective data strategy encompassing the collection, processing, management, analysis, and visualization of monitoring data sets to support decision-making, and the establishment of a cost-effective and intelligent sensor network aligned with the objectives set through comprehensive communication with asset owners. Due to the high data rates and dense sensor installations, conventional processing techniques are inadequate for fulfilling monitoring functionalities and ensuring security. Cloud-computing emerges as a widely adopted solution for processing and storing vast monitoring data sets. Drawing from the authors' experience in implementing long-span bridge monitoring systems in the UK and China, this paper compares the advantages and limitations of employing cloud- computing for long-span bridge monitoring. Furthermore, it explores strategies for developing a robust data strategy and leveraging artificial intelligence (AI) and digital twin (DT) technologies to extract relevant information or patterns regarding asset health conditions. This information is then visualized through the interaction between physical and virtual worlds, facilitating timely and informed decision-making in managing critical road transport infrastructure.

In silico and in vivo study of anti-inflammatory activity of Morinda longissima (Rubiaceae) extract and phytochemicals for treatment of inflammation-mediated diseases.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditionally, the plant Morinda longissima Y.Z.Ruan (Rubiaceae) is used by ethnic people in Vietnam for the treatment of liver diseases and hepatitis. AIM OF THE STUDY: The study was designed to assess the efficacy of the 95% ethanolic extract of Morinda longissima roots (MLE) in experimental immune inflammation. The phytochemical variation of root extract and the chemical structures of natural compounds were also investigated using HPLC-DAD-HR-MS analysis. MATERIALS AND METHODS: Three different doses (100, 200, and 300 mg/kg b.w.) of MLE were chosen to determine anti-inflammatory activity. The mice were given orally extracts and monitored their behavior and mortality for 14 days to evaluate acute toxicity. The volume of the paw and the histopathological evaluation were carried out. The polyphenolic phytoconstituents of MLE extract were identified using LC/MS analysis. The anti-inflammatory efficacy in silico and molecular docking simulations of these natural products were evaluated based on their cyclooxygenase (COX)-1 and 2 inhibitory effects. RESULTS: This investigation showed the 95% ethanolic extract of Morinda longissima roots was found non-toxic up to 2000 mg/kg dose level in an acute study, neither showed mortality nor treatment-related signs of toxicity in mice. Eight anthraquinones and anthraquinone glycosides of Morinda longissima roots were identified by HPLC-DAD-HR-MS analysis. In the in vivo experiments, MLE was found to possess powerful anti-inflammatory activities in comparison with diclofenac sodium. The highest anti-inflammatory activity of MLE in mice was observed at a dose of 300 mg/kg body weight. The in silico analysis showed that seven out the eight anthraquinones and anthraquinone glycosides possess a selectivity index RCOX-2/COX-1 lower than 1, indicating that these compounds are selective against the COX-2 enzyme in the following the order: rubiadin-3-methyl ether < morindone morindone-6-methyl ether < morindone-5-methyl ether < damnacanthol < rubiadin < damnacanthol-3-O-ß-primeveroside. The natural compounds with the best selectivity against the COX-2 enzyme are quercetin (9), rubiadin-3-methyl ether (7), and morindone (4), with RCOX2/COX1 ratios of 0.02, 0.03, and 0.19, respectively. When combined with the COX-2 protein in the MD research, quercetin and rubiadin-3-methyl ether greatly stabilized the backbone proteins and ligands. CONCLUSION: In conclusion, the anthraquinones and ethanolic extract of Morinda longissima roots may help fight COX-2 inflammation. To develop novel treatments for inflammatory disorders linked to this one, these chemicals should be investigated more in the future.

The structural and mechanistic bases for the viral resistance to allosteric HIV-1 integrase inhibitor pirmitegravir.

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are investigational antiretroviral agents which potently impair virion maturation by inducing hyper-multimerization of IN and inhibiting its interaction with viral genomic RNA. The pyrrolopyridine-based ALLINI pirmitegravir (PIR) has recently advanced into Phase 2a clinical trials. Previous cell culture based viral breakthrough assays identified the HIV-1(Y99H/A128T IN) variant that confers substantial resistance to this inhibitor. Here, we have elucidated the unexpected mechanism of viral resistance to PIR. While both Tyr99 and Ala128 are positioned within the inhibitor binding V-shaped cavity at the IN catalytic core domain (CCD) dimer interface, the Y99H/A128T IN mutations did not substantially affect direct binding of PIR to the CCD dimer or functional oligomerization of full-length IN. Instead, the drug-resistant mutations introduced a steric hindrance at the inhibitor mediated interface between CCD and C-terminal domain (CTD) and compromised CTD binding to the CCDY99H/A128T + PIR complex. Consequently, full-length INY99H/A128T was substantially less susceptible to the PIR induced hyper-multimerization than the WT protein, and HIV-1(Y99H/A128T IN) conferred >150-fold resistance to the inhibitor compared to the WT virus. By rationally modifying PIR we have developed its analog EKC110, which readily induced hyper-multimerization of INY99H/A128T in vitro and was ~14-fold more potent against HIV-1(Y99H/A128T IN) than the parent inhibitor. These findings suggest a path for developing improved PIR chemotypes with a higher barrier to resistance for their potential clinical use.

Oligomeric HIV-1 Integrase Structures Reveal Functional Plasticity for Intasome Assembly and RNA Binding.

Integrase (IN) performs dual essential roles during HIV-1 replication. During ingress, IN functions within an oligomeric "intasome" assembly to catalyze viral DNA integration into host chromatin. During late stages of infection, tetrameric IN binds viral RNA and orchestrates the condensation of ribonucleoprotein complexes into the capsid core. The molecular architectures of HIV-1 IN assemblies that mediate these distinct events remain unknown. Furthermore, the tetramer is an important antiviral target for allosteric IN inhibitors. Here, we determined cryo-EM structures of wildtype HIV-1 IN tetramers and intasome hexadecamers. Our structures unveil a remarkable plasticity that leverages IN C-terminal domains and abutting linkers to assemble functionally distinct oligomeric forms. Alteration of a newly recognized conserved interface revealed that both IN functions track with tetramerization in vitro and during HIV-1 infection. Collectively, our findings reveal how IN plasticity orchestrates its diverse molecular functions, suggest a working model for IN-viral RNA binding, and provide atomic blueprints for allosteric IN inhibitor development.

The Drug-Induced Interface That Drives HIV-1 Integrase Hypermultimerization and Loss of Function.

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are an emerging class of small molecules that disrupt viral maturation by inducing the aberrant multimerization of IN. Here, we present cocrystal structures of HIV-1 IN with two potent ALLINIs, namely, BI-D and the drug candidate Pirmitegravir. The structures reveal atomistic details of the ALLINI-induced interface between the HIV-1 IN catalytic core and carboxyl-terminal domains (CCD and CTD). Projecting from their principal binding pocket on the IN CCD dimer, the compounds act as molecular glue by engaging a triad of invariant HIV-1 IN CTD residues, namely, Tyr226, Trp235, and Lys266, to nucleate the CTD-CCD interaction. The drug-induced interface involves the CTD SH3-like fold and extends to the beginning of the IN carboxyl-terminal tail region. We show that mutations of HIV-1 IN CTD residues that participate in the interface with the CCD greatly reduce the IN-aggregation properties of Pirmitegravir. Our results explain the mechanism of the ALLINI-induced condensation of HIV-1 IN and provide a reliable template for the rational development of this series of antiretrovirals through the optimization of their key contacts with the viral target. IMPORTANCE Despite the remarkable success of combination antiretroviral therapy, HIV-1 remains among the major causes of human suffering and loss of life in poor and developing nations. To prevail in this drawn-out battle with the pandemic, it is essential to continue developing advanced antiviral agents to fight drug resistant HIV-1 variants. Allosteric integrase inhibitors (ALLINIs) are an emerging class of HIV-1 antagonists that are orthogonal to the current antiretroviral drugs. These small molecules act as highly specific molecular glue, which triggers the aggregation of HIV-1 integrase. In this work, we present high-resolution crystal structures that reveal the crucial interactions made by two potent ALLINIs, namely, BI-D and Pirmitegravir, with HIV-1 integrase. Our results explain the mechanism of drug action and will inform the development of this promising class of small molecules for future use in antiretroviral regimens.

Emergence of Compensatory Mutations Reveals the Importance of Electrostatic Interactions between HIV-1 Integrase and Genomic RNA.

HIV-1 integrase (IN) has a noncatalytic function in virion maturation through its binding to the viral RNA genome (gRNA). Class II IN substitutions inhibit IN-gRNA binding and result in the formation of virions with aberrant morphologies marked by mislocalization of the gRNA between the capsid lattice and the lipid envelope. These viruses are noninfectious due to a block at an early reverse transcription stage in target cells. HIV-1 IN utilizes basic residues within its C-terminal domain (CTD) to bind to the gRNA; however, the molecular nature of how these residues mediate gRNA binding and whether other regions of IN are involved remain unknown. To address this, we have isolated compensatory substitutions in the background of a class II IN mutant virus bearing R269A/K273A substitutions within the IN-CTD. We found that the nearby D256N and D270N compensatory substitutions restored the ability of IN to bind gRNA and led to the formation of mature infectious virions. Reinstating the local positive charge of the IN-CTD through individual D256R, D256K, D278R, and D279R substitutions was sufficient to specifically restore IN-gRNA binding and reverse transcription for the IN R269A/K273A as well as the IN R262A/R263A class II mutants. Structural modeling suggested that compensatory substitutions in the D256 residue created an additional interaction interface for gRNA binding, whereas other substitutions acted locally within the unstructured C-terminal tail of IN. Taken together, our findings highlight the essential role of CTD in gRNA binding and reveal the importance of pliable electrostatic interactions between the IN-CTD and the gRNA. IMPORTANCE In addition to its catalytic function, HIV-1 integrase (IN) binds to the viral RNA genome (gRNA) through positively charged residues (i.e., R262, R263, R269, K273) within its C-terminal domain (CTD) and regulates proper virion maturation. Mutation of these residues results in the formation of morphologically aberrant viruses blocked at an early reverse transcription stage in cells. Here we show that compensatory substitutions in nearby negatively charged aspartic acid residues (i.e., D256N, D270N) restore the ability of IN to bind gRNA for these mutant viruses and result in the formation of accurately matured infectious virions. Similarly, individual charge reversal substitutions at D256 as well as other nearby positions (i.e., D278, D279) are all sufficient to enable the respective IN mutants to bind gRNA, and subsequently restore reverse transcription and virion infectivity. Taken together, our findings reveal the importance of highly pliable electrostatic interactions in IN-gRNA binding.

Crystallographic snapshots of ternary complexes of thermophilic secondary alcohol dehydrogenase from Thermoanaerobacter pseudoethanolicus reveal the dynamics of ligand exchange and the proton relay network.

Three-dimensional structures of I86A and C295A mutant secondary alcohol dehydrogenase (SADH) from Thermoanaerobacter pseudoethanolicus were determined by x-ray crystallography. The tetrameric structure of C295A-SADH soaked with NADP+ and dimethyl sulfoxide (DMSO) was determined to 1.85 Å with an Rfree of 0.225. DMSO is bound to the tetrahedral zinc in each subunit, with ligands from SG of Cys-37, NE2 of His-59, and OD2 of Asp-150. The nicotinamide ring of NADP is hydrogen-bonded to the N of Ala-295 and the O of Val-265 and Gly-293. The O of DMSO is connected to a network of hydrogen bonds with OG of Ser-39, the 3'-OH of NADP, and ND1 of His-42. The structure of I86A-SADH soaked with 2-pentanol and NADP+ contains (R)-2-pentanol bound in each subunit, ligated to the tetrahedral zinc, and connected to the proton relay network. The structure of I86A-SADH soaked with 3-methylcyclohexanol and NADP+ has alcohol bound in three subunits. Two of the sites have the alcohol ligated to the zinc in an axial position, with OE2 of Glu-60 in the other axial position of a trigonal bipyramidal complex. One site has 3-methylcyclohexanol bound noncovalently, with the zinc in an inverted tetrahedral geometry with Glu-60. The fourth site also has the zinc in a trigonal bipyramidal complex with axial Glu-60 and water ligands. These structures demonstrate that ligand exchange of SADH involves pentacoordinate and inverted zinc complexes with Glu-60. Furthermore, we see a network of hydrogen bonds connecting the substrate oxygen to the external solvent that is likely to play a role in the mechanism of SADH.

The crystal structure of the S154Y mutant carbonyl reductase from Leifsonia xyli explains enhanced activity for 3,5-bis(trifluoromethyl)acetophenone reduction.

Carbonyl reductase from Leifsonia xyli (LXCAR, UniProtKB - T2FLN4) can stereoselectively catalyze the reduction of 3,5-bis(trifluoromethyl)acetophenone (BTAP) to its corresponding alcohol, (R)-[3,5-bis(trifluoromethyl)phenyl]ethanol ((R)-BTPE), which is a valuable chiral intermediate for the synthesis of antiemetic drugs, Aprepitant and Fosaprepitant. Moreover, this protein was found to have a broad spectrum of substrate specificity and can asymmetrically catalyze the reduction of a variety of ketones and keto esters. Even though molecular modelling of this protein was done by Wang et al. (2014), a crystal structure has not yet obtained. In this study, a single mutant, S154Y, which was shown to have higher catalytic activity toward BTAP than that of the wild type, was overexpressed in Escherichia coli BL21 (DE3), purified, and crystallized. The crystal structure of LXCAR-S154Y explains how the mutant enzyme can work with BTAP more efficiently than wild type carbonyl reductase. Furthermore, the structure explains why LXCAR-S154Y can use either NADH or NADPH efficiently as a cofactor, as well as elucidates a proton relay system present in the enzyme.

High incidence of type 2 diabetes in a population with normal range body mass index and individual prediction nomogram in Vietnam.

AIMS: The study aimed at determining 5-year incidence and prediction nomogram for new-onset type 2 diabetes (T2D) in a middle-aged population in Vietnam. METHODS: A population-based prospective study was designed to collect socio-economic, anthropometric, lifestyle and clinical data. Five-year T2D incidence was estimated and adjusted for age and sex. Hazard ratio (HR) for T2D was investigated using discrete-time proportional hazards model. T2D prediction model entering the most significant risk factors was developed using the multivariable logistic-regression algorithm. The corresponding prediction nomogram was constructed and checked for discrimination, calibration and clinical usefulness. RESULTS: The age- and sex-adjusted incidence was 21.0 cases (95% CI: 12.2-40.0) per 1000 person-years in people with mean BMI of 22.2 (95% CI: 21.9-22.7 kg/m2 ). The HRs (95% CI) for T2D were 1.14 (1.05-1.23) per 10 mmHg systolic blood pressure, 1.05 (1.03-1.08) per 1 cm waist circumference, 1.40 (1.13-1.73) per 1 mmol/L fasting blood glucose, 1.77 (1.15-2.71) per sleeping time (<6 h/day vs 6-7 h/day) and 2.12 (1.25-3.61) per residence (urban vs rural). The prediction nomogram for new-onset T2D had a good discrimination (area under curve: 0.711, 95% CI: 0.666-0.755) and fit calibration (mean absolute error: 0.009). For the predicted probability thresholds between 0.03 and 0.36, the nomogram showed a positive net benefit, without increasing the number of false positives. CONCLUSION: This study highlighted an alarmingly high incidence of T2D in a middle-aged population with a normal range BMI in Vietnam. The individual prediction nomogram with decision curve analysis for new-onset T2D would be valuable for early detection, intervention and treatment of the condition.

Association of PD-L1 gene polymorphisms and circulating sPD-L1 levels with HBV infection susceptibility and related liver disease progression.

Soluble molecules of programmed death ligand 1 (sPD-L1) are known to modulate T-cell depletion, an important mechanism of hepatitis B virus (HBV) persistence and liver disease progression. In addition, PD-L1 polymorphisms in the 3'-UTR can influence PD-L1 expression and have been associated with cancer risk, although not definitively. The purpose of this study was to investigate the association of PD-L1 polymorphisms and circulating levels of sPD-L1 in HBV infection and live disease progression. In this study, five hundred fifty-one HBV infected patients of the three clinically well-defined subgroups chronic hepatitis B (CHB, n = 186), liver cirrhosis (LC, n = 142) and hepatocellular carcinoma (HCC, n = 223) and 240 healthy individuals (HC) were enrolled. PD-L1 polymorphisms (rs2297136 and rs4143815) were genotyped by in-house validated ARMS assays. Logistic regression models were applied in order to determine the association of PD-L1 polymorphisms with HBV infection as well as with progression of related liver diseases. Plasma sPD-L1 levels were quantified by ELISA assays. The PD-L1 rs2297136 AA genotype was associated with HBV infection susceptibility (HBV vs. HC: OR = 1.6; 95%CI = 1.1-2.3; p = 0.0087) and disease progression (LC vs. CHB: OR = 1.8; 95%CI = 1.1-2.9; p = 0.018). Whereas, the rs2297136 GG genotype was a protective factor for HCC development. Plasma sPD-L1 levels were significantly high in HBV patients (p < 0.0001) and higher in the LC followed by CHB and HCC groups. High sPD-L1 levels correlated with increased liver enzymes and with advanced liver disease progression (Child-pugh C > B > A, p < 0.0001) and BCLC classification (BCLC D > C > B > A, p = 0.031). We could, for the first time, conclude that PD-L1 rs2297136 polymorphism and plasma sPD-L1 protein levels associate with HBV infection and HBV-related liver disease progression.

A simple nomogram for identifying individuals at high risk of undiagnosed diabetes in rural population.

AIMS: To sought for an easily applicable nomogram for detecting individuals at high risk of undiagnosed type 2 diabetes. METHODS: The development cohort included 2542 participants recruited randomly from a rural population in 2011.The glycemic status of subjects was determined using the fasting plasma glucose test and the oral glucose tolerance test. The Bayesian Model Average approach was used to search for a parsimonious model with minimum number of predictor and maximum discriminatory power. The corresponding prediction nomograms were constructed and checked for discrimination, calibration, clinical usefulness, and generalizability in nationwide population in 2012. RESULTS: The non-lab nomogram including waist circumference and systolic blood pressure was the most parsimonious with the area under receiver operating characteristic curve (AUC) of 0.71 (95 %CI = 0.64-0.76). Adding low-density lipoprotein cholesterol in the non-lab nomogram generated the lab-based nomogram with significantly improved AUC of 0.83 (0.78-0.87, P < 0.001). The nomograms had a positive net benefit at threshold probability between 0.01 and 0.15. Applying the non-lab nomogram to the national population yielded the AUC of 0.66 (0.63-0.70) and 0.68 (0.65-0.71) in the cohorts aged 40-64 and 30-69 years, respectively. CONCLUSIONS: The novel nomograms could help promote the early detection of undiagnosed diabetes in rural Vietnamese population.

Dammarane triterpenes and phytosterols from Dysoxylum tpongense Pierre and their anti-inflammatory activity against liver X receptors and NF-κB activation.

Dysoxylum tpongense Pierre (local name 'Huynh Dan Bap') belonging to family Meliaceae, is a tree (3-10 m height), distributed in the mountainous areas (ca. 1000 m a.s.l.) in North Vietnam. From the dichloromethane fraction of the methanol extract of the leaves and stems of this plant, six dammarane triterpenes, one furanoid diterpene together with three sterols were isolated. Evaluation of biological activities of isolated compounds showed that cabraleahydroxylactone (5), cabraleahydroxylactone 3-acetate (6), and stigmast-4-en-3-one (10) possessed an anti-inflammatory effect against Liver X receptor (LXR) activation in HepG2 cell line model with IC50 values of 20.29 ± 3.69, 24.32 ± 2.99, and 7.09 ± 0.97 (µM), respectively. While three other triterpenoid compounds aglinin C 3- acetate (1), aglinin C (2), and 24-epi-cabraleadiol (4) presented the most significant inhibitory effect against TNF-α induced NF-κB activation in HepG2 cell line in a dose-dependent manner with IC50 values of 12.45 ± 2.37, 23.32 ± 3.25, and 13.95 ± 1.57 µM, respectively. As stigmast-4-en-3-one (10), with structure closely similar to cholesterol, acted selectively on LXRs but not on NF-kB activation pathway, this suggests that stigmast-4-en-3-one (10) can be potentially applied as an agonist on LXR signaling pathway. Pathways LXRs-NF-κB-iNOS expression have a close relationship and play a crucial role in proceeding metabolic abnormalities like atherosclerosis, obesity, inflammation, etc. Thus, the findings showed that dammarane-type triterpenoids from D. tpongense are worthy of further investigation for potential LXR agonists and potent anti-atherogenic agents against atherosclerotic lesion progression.

Design and Implementation of a New System for Large Bridge Monitoring-GeoSHM.

Structural Health Monitoring (SHM) is a relatively new branch of civil engineering that focuses on assessing the health status of infrastructure, such as long-span bridges. Using a broad range of in-situ monitoring instruments, the purpose of the SHM is to help engineers understand the behaviour of structures, ensuring their structural integrity and the safety of the public. Under the Integrated Applications Promotion (IAP) scheme of the European Space Agency (ESA), a feasibility study (FS) project that used the Global Navigation Satellite Systems (GNSS) and Earth Observation (EO) for Structural Health Monitoring of Long-span Bridges (GeoSHM) was initiated in 2013. The GeoSHM FS Project was led by University of Nottingham and the Forth Road Bridge (Scotland, UK), which is a 2.5 km long suspension bridge across the Firth of Forth connecting Edinburgh and the Northern part of Scotland, was selected as the test structure for the GeoSHM FS project. Initial results have shown the significant potential of the GNSS and EO technologies. With these successes, the FS project was further extended to the demonstration stage, which is called the GeoSHM Demo project where two other long-span bridges in China were included as test structures. Led by UbiPOS UK Ltd. (Nottingham, UK), a Nottingham Hi-tech company, this stage focuses on addressing limitations identified during the feasibility study and developing an innovative data strategy to process, store, and interpret monitoring data. This paper will present an overview of the motivation and challenges of the GeoSHM Demo Project, a description of the software and hardware architecture and a discussion of some primary results that were obtained in the last three years.

Movement and contact patterns of long-distance free-grazing ducks and avian influenza persistence in Vietnam.

Presence of ducks, and in particular of free-grazing ducks, has consistently been shown to be one of the most important risk factors for highly pathogenic avian influenza outbreaks which has compromised poultry production in South-East Asia since the early 2000s and continues to threaten public health, farmers' livelihood and food security. Although free-grazing duck production has been practised for decades in South-East Asia, there are few published studies describing this production system, which is suspected to play an important role in the maintenance of avian influenza viruses. This study aimed at describing quantitatively the long-distance free-grazing duck production system in South Vietnam, characterising the movement and contact patterns of the duck flocks, and identifying potential associations between farming practices, movement and contact patterns and the circulation of avian influenza viruses. We conducted interviews among stakeholders involved in the free-grazing duck production system (duck farmers, transporters and rice paddy owners) in combination with a virological cross-sectional survey in South Vietnam. Results show that both direct and indirect contacts between free-grazing duck flocks were frequent and diverse. The flocks were transported extensively across district and province boundaries, mainly by boat but also by truck or on foot. A third of the investigated flocks had a positive influenza A virology test, indicating current circulation of avian influenza viruses, but none were positive for H5 subtypes. The age and size of the flock as well as its location at the time of sampling were associated with the risk of influenza A circulation in the flocks. These findings should be considered when developing risk assessment models of influenza virus spread aimed at informing the development of improved biosecurity practices leading to enhanced animal health, sustainable animal production and reliable income for farmers.

Dual involvement of G-substrate in motor learning revealed by gene deletion.

In this study, we generated mice lacking the gene for G-substrate, a specific substrate for cGMP-dependent protein kinase uniquely located in cerebellar Purkinje cells, and explored their specific functional deficits. G-substrate-deficient Purkinje cells in slices obtained at postnatal weeks (PWs) 10-15 maintained electrophysiological properties essentially similar to those from WT littermates. Conjunction of parallel fiber stimulation and depolarizing pulses induced long-term depression (LTD) normally. At younger ages, however, LTD attenuated temporarily at PW6 and recovered thereafter. In parallel with LTD, short-term (1 h) adaptation of optokinetic eye movement response (OKR) temporarily diminished at PW6. Young adult G-substrate knockout mice tested at PW12 exhibited no significant differences from their WT littermates in terms of brain structure, general behavior, locomotor behavior on a rotor rod or treadmill, eyeblink conditioning, dynamic characteristics of OKR, or short-term OKR adaptation. One unique change detected was a modest but significant attenuation in the long-term (5 days) adaptation of OKR. The present results support the concept that LTD is causal to short-term adaptation and reveal the dual functional involvement of G-substrate in neuronal mechanisms of the cerebellum for both short-term and long-term adaptation.

Value of ultrasound in the early diagnosis of prerupture uterine horn pregnancy. A case report.

BACKGROUND: The poor outcome of rudimentary horn pregnancies is due to delayed diagnosis. CASE: Pregnancy in a rudimentary uterine horn was detected by ultrasound prior to rupture. The rudimentary horn was resected without complications. CONCLUSION: Ultrasonography is essential in the early diagnosis of pregnancy in a rudimentary horn.

Atypical glandular cells: new Bethesda Terminology and Management Guidelines.

Although Pap tests have enabled early detection of premalignant lesions, the introduction of new collecting devices has significantly improved the detection of lesions hidden in the endocervical canal, such as adenocarcinoma in situ (AIS). The term "atypical glandular cells of undetermined significance" (AGUS) was introduced at the 1988 Bethesda Conference and defined as morphologic changes in glandular cells beyond those that are suggestive of the benign reactive process, but insufficient for the diagnosis of adenocarcinoma in situ (AIS). In the new 2001 Bethesda System, the term has been eliminated and replaced with the term "atypical glandular cells" (AGC), with the following subclassifications: not otherwise specified (NOS), favor neoplasia, endocervical AIS, and adenocarcinoma. The risks of premalignant or malignant disease associated with the AGC favor neoplasia category are substantially higher than in the AGC NOS category (96% vs. 9-41%, respectively). Patients diagnosed with AGC NOS or AGC favor neoplasia will require colposcopy, endocervical sampling, and, for patients over 35 years of age, endometrial biopsy. If all of these tests are negative, the Pap test should be repeated in 4-6 month intervals until 4 consecutive normal tests are obtained. Positive results in one of the tests will require management according to ASCCP guidelines. The AGC favor neoplasia diagnosis also requires cervical conization and/or other testing, as the incidence of premalignant or malignant lesions in patients with this diagnosis is high. The role of HPV testing in this setting is unknown at this time.

Management of atypical glandular cells of undetermined significance pap smears: a reappraisal.

OBJECTIVE.: To find appropriate management of patients with atypical glandular cells of undetermined significance (AGUS) Pap smears. MATERIALS AND METHODS.: The authors present their findings on patients with 606 AGUS Pap smears based on colposcopy, cervical biopsies, endocervical curettage, and endometrial biopsy. Twenty-one proposed management schemes by other authors are reviewed. RESULTS.: From 606 AGUS Pap smears, 69% had benign findings and 31% had neoplastic findings. Among 29 patients with endometrial pathology, only 4 patients (14%) were <40 years old. All proposed management schemes were uniform in the use of colposcopy but differed on the selective use of endometrial biopsy. CONCLUSION.: An AGUS Pap smear requires colposcopy. We suggest endometrial biopsy for women over 40 years old and for women younger than 40 years old who have a high risk for endometrial neoplasia. Better-defined cytologic criteria from cytologists are needed.

Documentation of amniotic fluid embolism via lung histopathology. Fact or fiction?

OBJECTIVE: To evaluate pulmonary histopathology for confirming amniotic fluid embolism. STUDY DESIGN: The Capra hircus (goat) model with fresh, homologous amniotic fluid was used. Raw fluid (n = 8), fluid filtered through a 5-microns filter (n = 14) and meconium-stained fluid with 1-7% solid debris (n = 7) were injected. Three hours after embolization the animals were euthanized and specimens collected. Three to five areas of lung were sampled based on the most abnormal areas visually. Traditional and special stains were utilized. The study protocol was approved by the institutional review board and animal use and care committee. Statistical analysis was by chi 2 with Yates correction. Significance was defined as P < .05. RESULTS: Amniotic fluid debris (fetal squames, mucin or foreign pigments) was found in 10 of 29 animals (34.5%). Debris was found in 7/7 (100%) of the meconium group, 2/8 (25%) of the raw fluid group and 1/14 of the filtered group (7%). The likelihood of finding debris in amniotic fluid embolism with meconium-stained fluid was greater than with raw (P < .017) or filtered amniotic fluid (P < .001). CONCLUSION: In this animal model, histopathologic confirmation of amniotic fluid embolism was an unreliable marker of the event except in cases of amniotic fluid embolism involving meconium-stained fluid.