Early initiation of antiviral therapy in elderly patients with influenza is associated with reduced risk of extra clinic visit, hospitalization and death. This study examined the cost-effectiveness of molecular POCT for detection of influenza viruses in Hong Kong elderly patients with influenza-like illness (ILI) in the outpatient clinics.A decision analytic model was used to simulate outcomes of a hypothetical cohort of elderly patients presented with ILI at outpatient clinics during peak season of influenza with two diagnostic approaches: Rapid molecular assay (POCT-PCR group) and clinical judgement with no POCT. Outcome measures included influenza-associated direct medical cost, hospitalization and mortality rates, quality-adjusted life year loss (QALY loss), and incremental cost per QALY saved (ICER).In base-case analysis, POCT-PCR group was expected to reduce hospitalization (1.38% versus 2.85%) and mortality rate (0.08% versus 0.16%) and save 0.00112 QALYs at higher cost (by USD33.2 per ILI patient), comparing with clinical judgement group. The ICER of POCT-PCR was 29,582 USD/QALY saved. One-way sensitivity analyses found ICER sensitive to: Hospitalization rate without prompt antiviral therapy; odds ratio of hospitalization with prompt therapy; influenza prevalence; patient age and mortality rate of hospitalized patients. POCT-PCR was cost-effective in 60.6% and 99.4% of 10,000 Monte Carlo simulations at willingness-to-pay threshold of 1x and 3x gross domestic product per capita of Hong Kong, respectively.Molecular POCT for influenza detection in elderly patients with ILI at outpatient clinics during peak influenza season appeared to be cost-effective in Hong Kong. The Hong Kong healthcare system is burdened by seasonal influenza every year. Influenza-associated complications lead to excess hospitalizations and deaths. In 2016, the Centre for Health Protection of Hong Kong reported in the sentinel surveillance that the annual incidence of influenza-like illness (ILI) was 30–94 per 1,000 cases at outpatient clinics and 110–300 per 1,000 cases at the accident and emergency departments [Early initiation of neuraminidase inhibitor therapy in patients with influenza is associated with reduced risk of extra clinic visit, hospitalization and death . The popA decision tree model  was desiIn both arms, patients presented with ILI might or might not be infected with influenza. In the POCT-PCR arm, patients were tested using rapid PCR molecular assay. A full course of oseltamivir was prescribed to patients who were tested positive for influenza, and symptomatic treatment for those who were tested negative. In the clinical group, the initiation of oseltamivir was based solely upon clinical judgement.After the index clinic visit, the patients infected with influenza might not seek further medical care, seek additional outpatient care, or be hospitalized. Those patients who were admitted to inpatient care might or might not receive care in the intensive care unit, and might survive or die.A literature search on MEDLINE over the period of 2000–2017 was performed using the keywords “influenza infection”, “neuraminidase inhibitor”, “oseltamivir”, “antiviral therapy”, “early treatment”, “outpatient visit”, “hospitalization”, “mortality” and “utility score”. The selection criteria of clinical studies of influenza infection were: (1) reports written in English; (2) etiology of ILI was identified as seasonal influenza infection, and (3) mortality rate and/or ICU admission rate were reported. All articles retrieved were screened for relevance to our model. For variable that was reported in multiple studies, the weighted average was used to estimate the base-case value and high/low values of the variable formed the range of sensitivity analysis. Subgroup data on patients aged 60 years or above from identified clinical reports were used for estimation of model inputs. When elderly subgroup data was not available, extended sensitivity analysis was performed on the non-age specific parameter by broadening the range of value.Model inputs were shown in The prevalence of influenza among patients presented with ILI during peak season was estimated from the weekly statistics on detection of influenza viruses from respiratory specimens reported by the Centre for Health Protection in 2016 . The weeThe QALY loss for influenza infection was calculated using the disutility , and the duration of time-spent in each of following events: (1) outpatient care; 2) hospitalization without ICU admission; (3) ICU care; and (4) influenza-related death. The utility value of each health status was estimated from findings of health-related quality of life studies –16. The  hospitalThe economic analysis of direct medical cost was conducted from the perspective of healthcare provider in Hong Kong. Cost items included rapid molecular test, oseltamivir , symptomatic treatment, outpatient clinic visit, hospitalization with and without ICU care. The cost per rapid molecular test  for influenza was adopted from literature . The cosPOCT-PCR − Costclinical judgement)/. As recommended by the World Health Organization (WHO), ICER less than 1x gross domestic product (GDP) per capita was considered as highly cost-effective and ICER less than 3x GDP per capita was cost-effective. The GDP per capita of Hong Kong was USD43,497 in 2016 [Expected value of each outcome measure  was simulated with the base-case model input values. Comparing with clinical group, if the POCT-PCR group saved QALYs at higher cost, incremental cost of per QALY saved (ICER) would be calculated using the following equation:  and Microsoft Excel 2010  to examine the robustness of the model results. The range for sensitivity analysis was the 95% CI range, high/low values of the variable or +/- 20% of the base-case value. One-way sensitivity analysis on all model inputs was performed to identify influential factors with threshold value.Some model inputs were not age-specific to elderly patients or Hong Kong practice, and extended sensitivity analysis was conducted on such parameters with broadened ranges. The sensitivity and specificity of clinical judgement were retrieved from a study conducted in the US . VariatiTo evaluate the impact of uncertainty in all variables simultaneously, a probabilistic sensitivity analysis was performed using Monte Carlo simulation. The direct cost and QALY loss of POCT-PCR and clinical judgement arms were recalculated 10,000 times by randomly drawing each of the model input from a triangular probability distribution within the range for sensitivity analysis. The probability of each study arm to be the preferred option was determined over a wide range of 1-3x GDP per capita as threshold of WTP.The base-case analysis results were shown in One-way sensitivity analysis found that the POCT-PCR group remained QALY-saving at higher cost throughout variation of all model inputs over the range for sensitivity analysis. To examine the impact of lowering cost per rapid-PCR test on the total cost, the range for sensitivity analysis (USD38-58) was extended to USD10-58. The extended one-way sensitivity analysis found a threshold value that the total cost in POCT-PCR was lower than clinical judgement group when the cost per rapid molecular assay was <USD14.8.The impact of one-way variation of all parameters on ICER of POCT-PCR was shown in a tornado diagram . The accThe probabilistic sensitivity analysis was performed by 10,000 Monte Carlo simulations. A scattered plot  showed tThe probabilities of POCT-PCR group to be the preferred option were examined in the acceptability curve over a wide WTP range (0 to 3x GDP per capita) . Using 1Our study investigated the cost-effectiveness of applying rapid molecular assay as POCT for detecting influenza at the ambulatory care setting. The base-case findings showed that POCT-PCR saved QALYs by reducing the rates of subsequent hospitalization for influenza and mortality. The POCT-PCR incurred higher total direct cost when compared to the clinical judgement group by USD33.2 per patients tested. The expected ICER of POCT-PCR was USD29,582, lower than 1x GDP per capita of Hong Kong . The base-case ICER was therefore highly cost-effective from the perspective of healthcare provider in Hong Kong.The one-way sensitivity analysis found the base-case results on QALYs saved and incremental cost incurred by POCT-PCR to be robust. POCT-PCR remained to save QALYs at higher cost than the clinical judgement group throughout variation of all model parameters. The acceptance of POCT-PCR to be highly cost-effective (ICER <1x GDP per capita) was sensitivity to variation of five influential factors. Four of the five factors were model inputs on outcomes of hospitalization for influenza: Hospitalization rate without prompt initiation of oseltamivir, odds ratio of hospitalization with prompt oseltamivir, mortality rate in hospitalized patients, and age. These four factors represented the effectiveness of prompt initiation oseltamivir (as a result of accurate diagnosis of influenza in the early state of infection) in prevention of hospitalization. The effectiveness in reduction of hospitalization for influenza was highly influential on the POCT-PCR cost-effectiveness. The costs per hospitalized case of seasonal influenza in elderly with and without ICU were reported to be substantial in Hong Kong , and theA health economic study compared the direct medical costs of applying in-house rapid PCR test for detecting influenza in patients with ILI at the emergency department and in hospitalized patients . The finInfluenza vaccine is the recommended measure for prevention of influenza, yet the vaccination coverage in Hong Kong elderly (39.1%) has been lower than optimal . Near-paThe present study was limited by intrinsic limitations of decision-analytic modeling that the model results were subject to uncertainties of model inputs. Sensitivity analyses were therefore conducted by one-way variation of each input and simultaneous variation of all parameters to examine the robustness of base-case results. The real-life events of ILI and influenza infection were simplified in the model. Different rapid PCR assays are categorized to various levels of technical complexity. The accuracy of technical skills in conducting the POCT-PCR assay might be associated with the level of product complexity, affecting the transferability of POCT-PCR sensitivity and specificity from clinical trial to daily practice. Variation of medical training programs in different countries also limited the transferability of overseas sensitivity and specificity of clinical judgment to Hong Kong. The cost and QALY loss in clinical judgment arm might be overestimated in some false-negative cases which are sub-clinical types of influenza with low hospitalization rate. The transmission of infection from influenza patients to uninfected subjects and misused of antibiotics for influenza patients were not included. The economic benefits and QALYs saved by early detection and treatment of influenza using POCT-PCR might therefore be underestimated.In conclusion, using rapid molecular assay as POCT for detection of influenza in elderly patients with ILI (for less than 7 days) at outpatient clinics during period with high circulation of influenza viruses appeared to be a cost-effective option to reduce hospitalization and mortality rate, and save QALYs from the perspective of healthcare provider in Hong Kong. The cost-effectiveness of POCT-PCR was subject to the threshold of WTP, age of elderly, prevalence of influenza, baseline hospitalization rate without prompt antiviral therapy, effectiveness of prompt initiation of antiviral therapy to reduce hospitalization, mortality rate of hospitalized elderly for influenza, and accuracy of clinical judgement and PCR for influenza.


Reconstructing the extent, flow and decay of the Laurentide Ice Sheet (LIS) on the continental shelves of North America during the last glaciation provides paleoglaciological analogues that are essential for understanding and predicting how modern marine-based ice-sheets will respond to future climate change and sea level fluctuations. The geometry of the LIS during Marine isotope stage 2  is one key element for ice-sheet modelling. The maximum extent of the LIS during this stage is well constrained for most sectors of the ice sheet, but major uncertainties remain, especially along the continental shelves of Arctic Canada. Despite a series of recent papers, the extent of the LIS in Western Baffin Bay, an area draining large volumes of glacial ice through multiple ice streams and likely characterized by ice shelves, remains highly speculative. Here we present unequivocal marine geophysical evidence that during the MIS-2 the LIS extended to the edge of the continental shelf, seaward of the previously proposed limits and subsequently retreated episodically westward during deglaciation. These data support interpretations of deep glacial ice grounding in Western Baffin Bay. The Flint (extensive) scenario1 was supported by modelling4 but, however, lacked field data while the minimalist scenario has been challenged by recent dating of moraines6 and by the identification of terrain preserved under cold-based ice on coastal forelands8. Cosmogenic nuclides exposure dating on terrestrial moraines as well as lacustrine and marine sediment cores data were used to resolve the enigma of the maximum extent of the LIS by proposing a scenario where most of LIS outlet glaciers reached the continental shelf at fjord mouths, with ice margins terminating inland in-between fjords 9. This interpretation was largely used in most paleogeographical reconstructions and in ice-sheet modelling studies15. More recent studies have, however, challenged this model, suggesting a more extensive LIS margin on the northeastern Baffin Island shelf during the Last Glacial Maximum (LGM) based on the observation of (1) an ice-contact seismostratigraphic unit extending on most of the continental shelf (the Baffin Shelf Drift16) and (2) a till wedge at 1300 m water depths at the southeast end of Lancaster Sound deposited at the ice grounding-zone17  are aligned in the trough axis and have low amplitudes (<20 m). Their very good state of preservation and the weak attenuation of their morphology due to a thin cover of postglacial sediments suggest that they relate to the last stage of ice occupation. Crosscut by grounding-zone wedges, MSGLs are interpreted to reflect time-transgressive ice-flows occurring during the landward retreat of an ice stream. In Pond and Scott troughs, MSGLs extend onto the shelf break  as the limit of the LIS margin during MIS-2.Four cross-shelf troughs that were glacially eroded and moulded during Quaternary glaciationsghs Fig. . These meak Figs . A smalleak Figs . These l24 which restricts vertical accommodation space for sediments in favor of sediment progradation, leading to the formation of low-amplitude and horizontally extensive landforms 23. GZWs are observed in the four studied troughs from swath bathymetry imagery and/or seismic data  that is coherent with postglacial sedimentation rates observed in Scott Trough16. Also, all the GZWs observed in Scott, Buchan and Pond troughs overprint sets of MSGLs formed during a previous phase of ice-flow  are formed by the accumulation of subglacial sediments at the grounding zone of an ice stream during temporary standstills of an ice margin e.g. ata Figs . On swatata Figs . On seisata Figs . The tra33. TMFs generally consist of stacked acoustically transparent lenses of glacigenic-debris flows (GDFs) separated by acoustically stratified units representing suspension-settling sediments, turbidites and/or contourites37. GDFs are mostly the product of instabilities at the grounding zone during the occupation of the trough by an ice stream, while the stratified units relate to deglacial to interglacial conditions43. Stacked GDFs interbedded with stratified units observed on seismic reflection profiles in Scott Trough could represent such a succession of glacial debris flows and interglacial sedimentation, but of unknown age age Fig. . At the ank Fig. . A hummoank Fig. . These l46. Multiple ISLMs are observed on both sides of Buchan and Scott troughs  on the outer continental shelf. Similarly to GZWs it could also indicate the presence of a constraint for vertical accumulation 47.Ice stream lateral moraines (ISLMs) indicate fast-flowing ice surrounded by either slower ice-flow or ice-free terrainghs Fig.  and 2, fghs Fig. . This di48. The chaotic acoustically semi-transparent unit extends laterally from the ridges and parallel to the shelf break; it is interpreted as ice-contact sediments correlated to the Baffin Shelf Drift16.Swath bathymetry imagery reveals ridges parallel to the shelf break on the banks seaward of Cape Adair Fig. . These r9 is located ~100 km eastward from the MSGLs terminus. Ice flowing through Pond Trough likely converged into the Lancaster Ice Stream in Bylot Trough and from there flowed eastward to reach the LGM grounding-zone on the Lancaster TMF17. Subglacial sediments delivered to Bylot Trough by the Pond Trough ice stream would have been taken in charge by Lancaster ice that fed the Lancaster Fan. In Sam Ford Trough, GZWs constructed from the same till/ice-contact seismic unit in both Buchan and Scott troughs extends at least to the middle of Sam Ford Trough to form a distinct transverse-to-flow ridge  the ice-contact sediments and the TMFs reported here could be of diachronic origin; (2)14C dates from these deposits are lacking; and (3) the LGM is a diachronic position corresponding to a time-period rather than a precise event in time. This time-period along eastern Baffin Island is yet to be resolved50.Ice-contact deposits observed at the shelf edge are interpreted as MIS-2 because (1) they occur down-ice (eastward) from series of GZWs deposited during an episodic westward retreat of the LIS margin; and (2) the grounding-zone wedges are overlain by only one sedimentary sequence of deglacial-postglacial deposition corresponding to the last glacial retreat. The thickness of the upper sedimentary sequence along with the sedimentation rates observed in Scott Trough support a deglacial origin for the GZWs. A deglacial age for the GZWs therefore implies a seaward position of the LIS margin during MIS-2. The LIS margin could have reached its maximum extent prior to the LGM53. The presence of cold-based ice in the inter-ice stream areas is in accordance with the preservation of older deposits than MIS-2 on coastal forelands in between the fjords7. The basal thermal regime of the LIS in the region seems therefore complex and spatially variable with low-gradient outlet fast-flowing ice being constrained to the fjords axis as proposed by Miller et al. (2002). In Sam Ford Trough, the absence of ice-flow morphologies and the presence of perpendicular-to-trough ridges suggest that ice-flow was slow in the trough. This slow ice-flow was probably due to the transport of ice discharge from Sam Ford Fjord into the Scott Inlet Ice Stream. The ice-flow route from Sam Ford Fjord to Scott Inlet can be inferred from the northward orientation of MSGLs and drumlins at the junction of Sam Ford Fjord and Hecla & Griper Trough. The location of the GZWs suggests that the retreat of the LIS margin from the shelf edge to the fjords head has been marked by several phases of stabilization.The revised ice-margin and the distribution of highly elongated bedforms (MSGLs) indicate that ice streams were operating along Buchan, Scott and Pond troughs during MIS-2. The presence of ISLMs suggests that the LIS was experiencing spatial variations in ice-flow velocities, suggesting that inter-trough areas, were submitted to slow ice-flow or cold-based ice50 and is in accordance with inferred LGM extents recently raised18.The swath bathymetry and geophysical data presented in this paper provide firm evidence that the LIS extended across the northeast Baffin Island continental shelf during MIS-2, reaching the continental shelf break between Scott Trough and Pond Trough. These data also confirm the presence of grounded glacial ice on the Lancaster Fan east of Pond Trough during MIS-2. The proposed maximum extent of the LIS during MIS-2 is located at least 36 km seaward of the previously proposed limits16. Finally, these results indicate that the LIS deglaciation was driven by a complex spatially varying thermal regime with low-gradient ice-streams in the troughs and cold-based/slow-flowing ice on the inter-trough areas. An LIS deglaciation from an outer seaward position implies differences in the LIS geometry, dynamics, flow-mechanisms and feedbacks to post-LGM relative sea-level rise in Western Baffin Bay. This revised ice margin and its retreat dynamics should therefore be taken into account to increase the accuracy of LIS reconstructions or modelling, which have implications on how we can understand the long-term evolution of a marine-based ice-sheet during deglaciation.Fast-flowing ice streams occupied Pond, Buchan and Scott troughs while slow ice-flow or cold-based ice occupied the shelf and Sam Ford Trough. The presence of GZWs in the four major cross-shelf troughs indicates an episodic retreat style punctuated by ice-margin stabilization rather than a rapid/catastrophic retreatwww.caris.com/products/hips-sips/) and MB-System (https://www.ldeo.columbia.edu/res/pi/MB-System/) softwares. Data visualization and mapping was realized using ESRI ArcGIS 10.4 software (http://www.esri.com). Seismic reflection data were analyzed and extracted using the LizardTech GeoViewer software (https://www.lizardtech.com/geoviewer-pro/overview). Maps and seismic reflection data were transferred to the Adobe Photoshop CS5 software (www.adobe.com/photoshop) for figure production and editing. Seismic reflection data were enhanced using the Brightness/Contrast tool in Adobe Photoshop CS5 for a clearer visualization.Multibeam data were collected using Kongsberg Simrad EM-300 (12 kHz) and EM-302 (30 kHz) echosounders onboard the CCGS Amundsen by the Ocean Mapping Group (University of New Brunswick) and the Laboratoire de Géosciences Marines  for the ArcticNet program. The multibeam data were processed using the Caris HIPS&SIPS (http://ftp.maps.canada.ca/pub/nrcan_rncan/raster/marine_geoscience/Seismic_Reflection_Scanned/).The multibeam bathymetry dataset can be visualized on the Université Laval Géoindex + website . The seismic reflection data along with the acquisition specifics are available on the Geological Survey of Canada website (


Microarray technologies inspired the development of carbohydrate arrays. Initially, carbohydrate array technology was hindered by the complex structures of glycans and their structural variability. The first designs of glycoarrays focused on the HTP (high throughput) study of protein–glycan binding events, and subsequently more in-depth kinetic analysis of carbohydrate–protein interactions. However, the applications have rapidly expanded and now achieve successful discrimination of selective interactions between carbohydrates and, not only proteins, but also viruses, bacteria and eukaryotic cells, and most recently even live cell responses to immobilized glycans. Combining array technology with other HTP technologies such as mass spectrometry is expected to allow even more accurate and sensitive analysis. This review provides a broad overview of established glycoarray technologies  and their emerging applications to the study of complex interactions between glycans and whole living cells. O-linked) or an asparagine residue (N-linked) depending on their distinct biosynthetic pathways. Proteoglycans represent a class of glycoconjugates richly dense in carbohydrates structures, which are generally long, unbranched molecules including glycosaminoglycans (GAGs), attached to a serine residue within the core protein via a xylose residue [Carbohydrates are a major group of biomolecules, which can be subdivided into smaller families of molecules categorized by their structures. In natural states, carbohydrates are found conjugated to other biomolecules including proteins and lipids. Glycolipids are carbohydrates covalently attached to lipids and consist of important constituents including glycosphingolipids (GSLs), diacylglycolipids (DAGs) and lipopolysaccharides (LPs). Glycoproteins are formed when carbohydrates are conjugated to proteins via a serine or threonine residue  in 2001, which is now a widely used web-resource aimed at providing researchers with data, tools, resources, and information about community activities in the growing field of functional glycomics.The use of microarrays for the study of carbohydrate interactions has lagged behind other biological molecules, with scientific literature appearing only in 2002 ,9. This de novo synthetic approaches. One example of the fundamental challenges of generating chemical libraries is the capacity to modify one specific hydroxyl group in the presence of many others. Strategies of glycan synthesis use steps to protect and mask chemical moieties in order to preferentially react the chemical groups of interest [One major consideration for the construction of glycan arrays is the source of the glycoconjugate used, which in itself determines the aim of the experimental strategy. Sources of glycoconjugates can be broadly divided into three main subgroups: (1) Naturally occurring structures, which are extracted and purified from biological sources. Drawbacks of this source are that purification of glycans are hampered with factors such as multiple experimental steps associated with sample losses at every stage and reagents and conditions that can change the original glycan structures . Methodsinterest . The secinterest . The useinterest . The useinterest . Translainterest . Automatinterest , which iinterest .Important factors to consider in the initial choice of solid supports for glycan arrays are twofold: whether derivatization of glycans is necessary and which type of chemistry is needed for immobilization of sugars onto the surface. However, this depends on the final experimental technique or the versatility of the resulting glycan array. Certain supports such as gold-based substrates provide advantages in that not only are they compatible for traditional fluorescence measurements using microarray scanners, but they can also be used as a platform for Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), surface plasmon resonance (SPR), and quartz crystal microbalances . The useThere are different types of immobilization methods for glycans which exploit the large structural variety of glycoconjugates see . One of One relatively straightforward method of immobilization of glycans onto a surface is adsorption. Glycans can be non-covalently and non-specifically immobilized onto nitrocellulose or oxidized black polystyrene surfaces ,31. NeogModification of glycan structures can be time-consuming and costly; therefore, methods of immobilization that can use unmodified glycans are sometimes preferable. This type of immobilization can be achieved with photoactivatable supports which contain photolabile groups such as aryl(trifluoromethyl)diazirine . Upon irThe localization of glycan immobilization can be assured by using methods that result in covalent site-specific attachment of glycans. Immobilization methods based on the reaction between thiol and malemide groups are an example of covalent site-specific attachment of glycans. Studies have used malemide-conjugated sugars immobilized onto thiol-derivatized surfaces ,43. ConvOne of the main disadvantages of covalent attachment to a monolayer is that high ligand concentrations are required which is somewhat quixotic when working with complex oligosaccharides . Glycan The choice of surface is important as it determines the type of detection that can be used, as the detection method depends on the fundamental properties of the surface. The most common type of detection method of carbohydrate arrays relies on fluorescence detection and the use of fluorescently labeled proteins binding directly or indirectly to the glycan structures. The standard mode of detection in this case is a fluorescent microarray scanner. Fluorescence detection can be achieved using different methodologies: (1) using proteins labeled with fluorophores, (2) using fluorophores as a secondary reagent or (3) using fluorescent proteins that bind tags. The latter two sandwich assay options can be used to increase signal amplification as the number of fluorophores available for detection is increased . DisadvaAs the cell membrane is a fluidic lipid bilayer environment, it is potentially important that the generation of glycan arrays mimic the lateral movement of glycans in their natural state at the cell membrane. Another important factor in protein–carbohydrate interactions is multivalency. Protein–carbohydrate interactions tend to be of low affinity but high specificity and so use multivalency to generate the affinity required for biologically relevant binding. In this way glycans can be organized to form clustered saccharide patches (CSPs) . Applicaet al. [Generic glycan arrays are available and allow the attachment of glycans from sources including mammals and microorganisms. One example of such a platform is described by Fukui et al. , which het al. .Antigen F77. The generation of designer NGLs probes from O-glycans was achieved by alkaline reductive release from a source of epithelial mucin, porcine stomach mucin (PSM). This method surprising found the F77 antigen to be expressed in blood group H on a 6-linked branch of a poly-N-acetyllactosamine backbone [d-glucose polymers with differing linkages in linear or branched sequences and function as secreted virulence factors in bacteria. By using combinatorial approaches involving negative-ion electrospray tandem MS, information was obtained on linkage sequence and chain length requirement of glucan-recognition proteins such as Dendritic Cell-Specific Intercellular Adhesion molecule-3-Grabbing Non Integrin (DC-SIGN) [In comparison, custom-made arrays are also designed to answer specific questions. One example is the inclusion of the “designer microarray” with other combinatorial approaches to define the carbohydrate sequence of the Prostate Cancer-associated backbone . AnotherDC-SIGN) . Other eDC-SIGN) ,57,58. AN-sulfated glucosamine (GlcNS) and one Ido2S containing disaccharide [-O-sulfate and the acetyl residue on an otherwise N-sulfated saccharide is responsible for heparin binding to increase the activity of antithrombin [Heparan sulfate proteoglycans (HSPGs) contain protein core proteins that are covalently attached with HS chains. HS is a ubiquitous linear polysaccharide molecule, belonging to the GAG family of macromolecules. Attachment of HS to different core proteins results in HS having the ability to alter its location and topography as core proteins can occur at the cell surface and the extracellular matrix . HSPGs accharide . Other ethrombin .3) and GlcNSO3(6-OSO3) residues is required [The minimal binding sequences are not always sufficient to increase biological activity. For FGF-2, twice the size of the minimum sequence is needed to produce a mitogenic response and a dodecasaccharide or longer sequence containing both IdoA  values for soluble inhibitors of proteins binding to glycans immobilized to surfaces. An example of the experimental setup includes a glycan array to which fluorescently labeled proteins are added, followed by soluble inhibitors. Following washing steps, the fluorescent intensities of the bound proteins are measured, which determines the IC50 values of the soluble inhibitor [One of the main first applications of glycan arrays was to study how glycan structure would affect protein binding properties. This is being studied using arrays of heparin structures. Libraries containing heparin structures varying in their degree of sulfation as well as the length of their saccharide chains have been generated using heparin depolymerization  techniquonstants . Studieseriments . The meanhibitor .N-acetylglucosamine (GlcNAc) residues, galactose  and N-acetylgalactosamine residues. Primary chicken hepatocytes express a well-defined C-type lectin that binds to non-reducing terminal N-acetylglucosamine residues, and was labeled with a fluorescent dye. A specific chamber was used to remove non-adherent cells  and adherent cells were measured using fluorescence detection. Chicken hepatocytes bound selectively to lectins derivatized with GlcNAc structures rather than spots of lectin with Gal or no modifications [Lectins are carbohydrate-binding proteins and macromolecules that are highly specific for sugar moieties. Lectins are present in plants, microbes and animals . Lectinsications .Glycan arrays have been very important in the understanding of surface interaction of various other microorganisms including interactions with viruses. The interaction of envelope glycoproteins with protein binding partners has been the basis of the development of the design of vaccines, which has been extensively exploited to interfere with the interaction involving the entry of the HIV virus. Glyco-protein microarrays have been used to analyze the glycan interactions dictating the interaction between the two HIV-1 (human immunodeficiency virus) glycoproteins, gp-120 and gp-41. The structures decorating the viral-surface envelope glycoproteins of HIV include high-mannose oligosaccharides including triantennary mannoside-1 . SynthesHelicobacter pylori and Escherichia coli. Initial interactions determine adherence, which is an important factor of pathogenicity in microorganisms and separate human microbiota from pathogenic bacteria [N-hydroxylsuccinimide (NHS) ester reactive group and so enable the covalent attachment of sugars [Carbohydrate–protein interactions also dictate interactions between human cells and other pathogens including various bacteria such as bacteria . Carbohybacteria . Immobilf sugars . After hf sugars . Therefof sugars .Candida albicans, which show the ability to bind aminoglycoside structures. In addition, differential binding can be detected by varying amounts of fluorescence.Carbohydrate arrays can also be used to characterize aminoglycoside antibiotics. Aminoglycosides contain aminocyclitol rings with one or more amino sugars. Aminoglycosides bind to various sites including the A site of 16S rRNA of bacteria as well as other RNA species ,87,88,890-G1-S phase progression due to regulation of cyclin D1. Mitogens such as FGF2 and particular HS saccharides structures in chlorate- treated Swiss 3T3 cells evoke a biphasic increase in ERK1/2 activity involved a large initial increase in activity followed by a degree of activity sustained at a lower level. This pathway could be studied in-depth by the development of a high-throughput (HTP) assay which allowed study of the functional effect of many HS structures simultaneously. 96-well plate assays have been used effectively in the past [et al. [The interactions of specific glycan structures with proteins may produce various functional consequences such as activation or inhibition of cellular processes. This means that functional assays are very important in addition to the protein interaction measurements; recent work has shown that glycoarrays can also be used to assess living cell responses to immobilized glycans. For example in the HS structure-function paradigm, many studies have analyzed the effects of HS structure on long-term cellular outcomes, such as cell proliferation, using standard cell biology techniques. Recently however, the direct investigation of the effect of different HS structures on cellular signaling in a glycoarray format has been described for the first time . Previouthe past . Puviraj [et al.  have des [et al. ,98. This2 and polylysine were arrayed onto the surface and then BaF3 cells in a viscous solution of alginate were then spotted onto the polylysine spots. Soluble growth factors and exogenous structures of HS and chondroitin sulfate (CS) were added to the media. The mechanism of FGF-FGFR-GAG signal transduction and how it relates to cellular activity can be examined by assessing cell proliferation using reagents which distinguish live cells from dead cells and assess intracellular esterase activity and plasma membrane integrity. Calcein AM and ethidium homodimer (EthD-1) dyes were used for this application [More recently, the study of molecular interactions dictating cellular processes such as cellular proliferation has now been made possible using 3D platforms and the use of 3D block printing. This has resulted in the development of high-throughput miniaturized 3D-chip platforms that can examine the importance of specific structures in HS and CS (chondroitin sulfate) in ternary structures of growth factor and growth factor receptor signaling complexes . 3D bloclication ,102,103.lication . A seconlication .Coupling mass spectrometry (MS) with glycan arrays provides a promising application for the discovery of new glycan-binding ligands and binding proteins. To date, the main achievements in this field have been made with respect to lectin microarrays. Applications include the systematic identification of carbohydrate-binding proteins in proteomes . One sucKey advances in glycan arrays over the past two decades have been in screening carbohydrate-binding proteins in proteomes, calculating protein binding affinities and automatization of solid-support synthesis for glycans. Integration and examination of this wealth of information is beginning to become more standardized with the use of public data repositories within consortia such as the CFG. Aspects of immobilization, choice of support and detection have now been studied for different types of glycans depending on the type of application. Technological developments mean that increased sensitivity as well as combinatorial approaches exploiting mass spectrometry techniques for glycan array interrogation will permit new methodological advances to continue. Further exploitation of glycobioarrays may also provide higher throughput functional level screening of glycan activities. Collectively, these will improve the quality and amount of data, plus improve quantitation of data. Ultimately, this will yield a wealth of insights into the functional diversity and functional specificity of glycans and will underpin new routes to exploit this knowledge in biomedical applications.


Youth who received RFCBT also demonstrated significant decreases in connectivity between the left PCC and the right inferior frontal gyrus (IFG) and bilateral inferior temporal gyri (ITG). Degree of change in connectivity was correlated with changes in self-report depression and rumination. These data suggest that rumination can be reduced over eight weeks and that this reduction is associated with parallel decreases in residual depressive symptoms and decreased functional connectivity of the left PCC with cognitive control nodes. These changes may enhance the ability of vulnerable youth to stay well during the transition to adulthood.This pilot randomized control trial was designed to examine whether Rumination-Focused Cognitive Behavior Therapy (RFCBT) reduces rumination and residual depressive symptoms among adolescents with a history of Major Depressive Disorder (MDD) who are at risk for relapse. We also examined whether these changes in symptoms were associated with changes in functional connectivity of the posterior cingulate cortex (PCC), a key node in the default mode network (DMN). Thirty-three adolescents (ages 12–18) were randomized to eight weeks of RFCBT or an assessment only (AO) control. Twenty two adolescents successfully completed fMRI scans pre- and post-intervention. Adolescents were recruited from the clinic and community and met criteria for at least one previous episode of MDD and were currently in full or partial remission. An Independent Evaluator interviewed parent and child before and after the eight-week intervention. The left PCC  seed was used to probe resting state functional connectivity of the DMN. Adolescents who received RFCBT demonstrated reduced rumination (F = -2.76, df = 112, Trial Registration: ClinicalTrials.gov NCT01905267 Major Depressive Disorder (MDD) with early onset can set youth on a trajectory towards lifelong illness and disability ,2. AlthoRumination, a thought pattern that involves passively and repetitively dwelling on negative feelings and their causes and consequences remains elevated following remission from depression and prospectively predicts the severity and duration of subsequent depressive episodes ,8. The rOne such intervention, Rumination-Focused Cognitive Behavior Therapy (RFCBT) has been tested as an adjunctive intervention to treatment-as-usual  among adults . In thisThe technology offered by functional magnetic resonance imaging (fMRI) allows for the examination of potential neural mechanisms in the longitudinal course of MDD . Indeed,For example, one study examined the RSFC of the subgenual anterior cingulate cortex (sgACC) among currently depressed adolescents in relation to rumination . IncreasAs such, the current investigation was designed to examine whether directly targeting rumination among a population of vulnerable adolescents in the remitted phase of MDD would be correlated with changes in the RSFC of the PCC. We hypothesized that RFCBT would reduce residual depression as well as reduce the habit of rumination and that these changes would be associated with changes in the RSFC of the PCC. Given the exploratory nature of the study, a data-driven approach was taken and connectivity of the PCC with the whole brain was examined.th edition [Participants were adolescents (n = 33) who met full Diagnostic and Statistical Manual of Mental Disorders, 4 edition  for MDD  edition ). Partia edition . Partici edition  < 70, a Adolescents randomized to RFCBT met with a therapist (first author) on a weekly basis for 45–60 minutes to receive the manualized intervention. The RFCBT intervention was adapted from materials designed for adults  to speciThe RADS  is a 30The 22-item RRS  was useThe CDRS-R  is a weThe K-SADS-PL  is a wehttp://www.fil.ion.ucl.ac.uk/spm/doc/) and motion detection algorithms were applied using FSL (http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/). Coregistration of structural images to functional images was followed by spatial normalization of the coregistered T1-spgr to the Montreal Neurological Institute (MNI) template. The resulting normalization matrix was then applied to the slice-time-corrected, physiologically corrected, time series data. These normalized T2* time-series data were spatially smoothed with a 5mm Gaussian kernel resulting in T2* images with isotropic voxels, 2mm on a side. Time series were detrended and mean centered. Physiologic correction was performed by regressing out white matter and cerebral spinal fluid signals [An eyes-open resting state scan was acquired over six minutes in a 3.0 T GE Discovery scanner  using parallel imaging with ASSET and T2* gradient-echo axial EPI with the following parameters: 90 degree flip, field-of-view 22, matrix size = 64x64, slice thickness = 3mm, 22.2ms echo time, 44 slices, with a TR of 2000 ms for a total collection of 200 TRs. Scan parameters were adjusted following the first several scans. High-resolution anatomic T1 scans were collected across four minutes with the following parameters: 13 degree flip, field-of-view 22, matrix size 256x256, slice thickness = 1mm, 182 slices. Motion was minimized with foam pads and a cross on the display, and by conveying the importance of holding still to participants. Several steps were taken to reduce potential sources of noise and artifact. Slice timing was completed with SPM8  normality plots of the average standard deviation of movement values in the x, y, and z planes were examined and those with values greater than 2 standard deviations (n = 3) were excluded, 2) individuals with any TR to TR movements greater than .5mm across three consecutive TRs were also excluded (n = 2). Combining these two methods resulted in stringent criteria for movement and a sample of 22 adolescents (11 in each group). There were no significant clinical or demographic differences between the fMRI sample and the full sample of 33 randomized adolescents and randomization was not related to movement status.p < .05 by using AlphaSim with 1000 Monte Carlo simulations to determine a joint threshold of height and extent . A full factorial second-level model including the effects of group and time was created. Given the current investigation was not adequately powered to detect treatment-by-time interactions; we chose to examine the main effect of time in the RFCBT group alone. To verify that these differences were related to treatment, we confirmed that all regions of interest (ROIs) that changed from baseline to week eight in the RFCBT group did not change over time in the AO group. Thus, only ROIs that changed in the RFCBT group and did not change in the AO group were evaluated.A 19 voxel sphere left PCC seed  was derived based on previous work examining PCC connectivity ,37 and pPost-hoc analyses evaluated relations between clinical symptoms and connectivity. Two-tailed Pearson correlations between change in depression and rumination (baseline to 8 week) and change in extracted connectivity values (scan one to scan two) were conducted.Mixed-effects regression models (MRMs) were conducted on the Intent-to-Treat (ITT) sample using SPSS MIXED. MRMs allow for the dependencies inherent in repeated assessments, are robust to missing data, and can be used to estimate scores using group trajectories. MRMs were used to assess the effects of treatment, and treatment-by-time , on the RRS, RADS, and CDRS-R across eight weeks. Clinical results based on the sample of treatment completers as well as the fMRI sample are presented in p < .01) and self-report depression across eight weeks  relative to adolescents in the AO condition. As illustrated in p = .08), wherein the AO group demonstrated a slight increase in symptoms compared to the RFCBT group who demonstrated a slight decrease. Supporting information including The main effect of time in the RFCBT group revealed significant changes in several regions. Increasing connectivity was observed from the left PCC to the postcentral (BA = 3) and fusiform (BA = 19) gyri, the thalamus, and the cerebellum.p < .01) and change in rumination .To reduce the number of statistical tests, correlations were run only with the two largest identified clusters: the right IFG and right ITG. Change in connectivity between the left PCC and right IFG was not significantly correlated with change in clinical measures. Change in connectivity between the left PCC and the right ITG was significantly correlated with change in self-report depression  and the right IFG (a node of the CCN) was reduced with RFCBT suggests that the DMN and CCN may begin to function more independently as skills for reducing rumination are learned. The IFG has been implicated in healthy emotion regulation  and receIt is noteworthy that the caudate has been highlighted as a region that may contribute to meditative states due to its role in attentional disengagement . DeliverOur finding that change in connectivity between the left PCC and right ITG was associated with change in depressive symptoms and change in rumination is somewhat surprising. The ITG is typically associated with semantic processing ; howeverOverall, the current findings build upon a recent review of fMRI studies among youth suffering from acute depression  that exaFrom a theoretical perspective, the original Response Styles Theory suggests that rumination is a stable trait . More reThe current study has several limitations. First, this was a small pilot study designed to examine feasibility, acceptability, and a preliminary test of a brain-based mechanism for a psychosocial intervention. Thus, the primary limitation of the current study is the sample size, particularly for the fMRI portion of the study. Future research adequately powered to fully examine treatment-by-time changes in connectivity is now warranted. In addition, the current sample is subject to the potential confound of ongoing psychosocial and medication treatment. Future research can evaluate the effectiveness of the intervention among a larger sample, including a population not currently receiving maintenance treatments such as psychiatric medication. A strength of the current design; however, is that it extends directly from the trial conducted with adults  which evRecurrent depressive episodes occur more frequently as illness progresses ; therefoS1 CONSORT Checklist(DOC)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S1 Study Protocol(DOCX)Click here for additional data file.S1 Supplement(DOCX)Click here for additional data file.


The renin-angiotensin-aldosterone system is acknowledged as being vital in the body's hormone system and plays a fundamental role in the maintenance of water and electrolyte balance and blood pressure regulation. There is growing evidence that the renin-angiotensin-aldosterone system has profound influences on the expression and bioactivity of BK channels. In this review, we focus on the molecular mechanisms underlying the regulation of BK channels mediated by the renin-angiotensin-aldosterone system and its potential as a target for clinical drugs.Large-conductance calcium-activated potassium channels (BK channels) belong to a family of Ca The renin-angiotensin-aldosterone system (RAAS) plays a key role in the maintenance of water and electrolyte balance and blood pressure regulation. In recent years, an increasing number of experiments have been conducted to explore the extremely complex relationship between BK channels and RAAS. This review provides an overview of the variety of mechanisms underlying the regulation of BK channels in different target tissues mediated by RAAS and its potential as a target for clinical drugs.Large-conductance calcium-activated potassium channels (BK channels) are expressed on cell membranes and involved in diverse physiological processes including controlling membrane potential and Ca+ channels, but differ in the mechanism of their activation  starting with an extracellular N-terminus and four hydrophobic regions (S7-S10) connecting with an intracellular C-terminus . S0 is a unique additional N-terminal transmembrane segment serving as a site of coupling with β-subunits; this functional interaction is responsible for modulating the balance between resting and active states of the VSD   that are encoded by genes 2+-dependent voltage-operated activation , is a crucial hormone system that contributes to maintaining water-electrolyte balance and regulating arterial blood pressure. BK channels are known to associate with several RAAS factors, with each of these interactions having distinct physiological consequences. The interaction between BK channels and RAAS is emerging as a key regulator of BK channel function in both health and disease.Renin, a protein and enzyme generated from cells in the kidney, plays a critical role in RAAS. It can cleave and activate another circulating protein, angiotensin, which represents the beginning of a chain reaction involving RAAS.Ang II, an octapeptide hormone, plays a major role in regulating the body's blood pressure and maintaining water-electrolyte balance through sympathetic nervous activity, aldosterone-regulated sodium excretion, and thirst responses  and regulate most of the physiological activities of Ang II in various kinds of target cells  phosphorylation is a primary mechanism through which Ang II exerts its function. The process leads to the activation of protein kinase C (PKC), a serine/threonine kinase  and protein kinase G (PKG), which results in the insensitivity of BK channels to the activation of PKG or PKA and subsequently inhibits cAMP-induced pulmonary vasodilatation  and peroxynitrite (OONO−), the latter causing BK channel tyrosine nitration. Ang II-induced oxidative post-translational modification makes it possible to impair BK channel function in vascular smooth muscle , which form part of an inactivator of Gα subunits, play a significant role in AT1R-mediated regulation of BK channel activity. Gβγ activates c-Src, which in turn activates Abelson murine leukemia viral oncogene homolog (c-Abl) tyrosine kinase. C-Src, a non-receptor tyrosine kinase, is abundant in vascular tissues and can be activated by ROS as well, thereby forming a self-perpetuating activation loop and encouraging sustained generation of oxidative stress in response to Ang II stimulation. AT1R interacts with Gα after stimulation by Ang II, which in turn activates PKC and enhances NOX-1 complex activity through neutrophil cytosol factor 1 phosphorylation and Ras-related C3 botulinum toxin substrate 1 phosphorylation. In the meantime, the combination of Ang II and AT1R interacts with Gβγ, which activates c-Src/c-Abl-dependent signaling pathway. The process leads to the phosphorylation of Cav-1 tyrosine 14 mediated by c-Abl, which facilitates the trafficking of the receptor into relatively cav1-enriched lipid rafts and contributes to the Ang II-related BK channel dysfunction in diabetes. ROS not only increases c-Src/protein tyrosine kinase (PTK) activity but also inhibits protein tyrosine phosphatase (PTP) activity, which results in an increase of the phosphorylation in protein tyrosine. BK channel cysteine oxidation is directly caused by hydrogen peroxide   is apparently elevated, which enhances K+, high K+ diet mice contributes to a high rate of BK channel-mediated potassium secretion, which leads to an osmotic diuresis. The high urinary flow created by BK-mediated K+ secretion may be activated in a positive feedback manner, indicating that BK channels may present a therapeutic target for treating hyperkalemia.Some investigators hold the view that the increase in aldosterone may enhance the activity of BK channels under conditions of high dietary potassium intake. Cornelius et al.  tend tow+ excretion is associated with a kaliuretic pathway, rather than aldosterone directly affecting renal potassium ion transport due to changes in plasma potassium concentration. Recent studies have revealed a new understanding of the major role of BK channels activated by aldosterone-independent kaliuretic factors in which BK channel expression levels are closely related with flow-stimulated K+ secretion (Woda et al., + excretion is deemed to be dependent on tubular flow rate and BK channels are activated by an increase in the tubular flow rate (Woda et al., 2+ concentration, which enables BK channels to translocate from the cell to the apical membrane. Through further studies, they found that PKA and PKC are involved in regulating the activity of BK channels in the mammalian distal nephron (Liu et al., In contrast, some studies deem that aldosterone has little or no influence on the expression or the activity of BK channels in response to dietary potassium intake. Estilo et al.  hold theACE inhibitors, which constitute a wide range of important clinical targeted drugs for RAAS, are a class of clinical drugs used mainly for the treatment of congestive heart failure and hypertension. Commonly prescribed ACE inhibitors include captopril, enalapril, zofenopril, perindopril, trandolapril, ramipril, and lisinopril. This group of drugs inhibits angiotensin-converting enzyme, an important component of the RAAS, which leads to hypovolemia as well as vasodilatation and causes decreased blood pressure and heart oxygen requirement. Albarwani et al.  found thIn summary, RAAS is deemed to have great influence on the expression and bioactivity of BK channels to regulate its physiological function via multiple mechanisms mediated by different components in this system. Ang II possesses a dual-aspect function that acts on BK channels after binding with the two types of receptors, AT1R and AT2R. The aldosterone paradox is a recently proposed concept in which aldosterone leads to two distinct physiological effects under conditions of hypovolemia and hyperkalemia to maintain the fluid and electrolyte balance. To some extent, this indicates that aldosterone-mediated regulation of BK channels is a very complex biological process, rather than a straightforward one mediated by a single mechanism. ACE inhibitors, such as the clinical commonly used RAAS-targeted drugs, contribute to the beneficial effects mediated by the endothelium on the cardiovascular system. There are conflicting study results as to whether the process is related to the decreased activity of BK channels induced by ACE inhibitors. Hence, more in-depth studies are required in the future, with any novel findings applied to developing new clinical treatments.ZZ wrote the initial drafts. LQ and RW revised the review and finalized the last version of the article. All authors checked and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The distribution of Extended-Spectrum Beta-Lactamase-producing Gram-negative bacteria (ESBL-GNB) colonization sites is relevant for infection control guidelines on detection and follow-up of colonization. We questioned whether it is possible to rely solely on rectal swab culture for follow-up of ESBL-GNB colonization.We retrospectively assessed ESBL-GNB colonization sites in patients in a tertiary hospital in the Netherlands. The Laboratory Information Management System was queried for all bacterial cultures obtained between January 2012 and August 2016. All patients with one or more cultures positive for ESBL-GNB were identified and the distribution of ESBL-GNB positive sample sites was assessed. A subgroup analysis was performed on patients for whom at least one rectal swab specimen was available.We identified 1011 ESBL-GNB carriers with 16,578 specimens for analysis. ESBL-GNB were most frequently isolated from the rectum (506/1011), followed by the urogenital (414/1011) and respiratory tract (142/1011), and pus (136/1011). For 588 patients at least one rectal swab specimen was available. In this subgroup, ESBL-GNB colonization was detected only in the rectum in 55.4% (326/588) of patients, in 30.6% (180/588) in the rectum and a different culture site, and in 13.9% (82/588) no rectal colonization was detected.Rectal colonization with ESBL-GNB was detected in 86% of ESBL-GNB carriers. However, in 14% of ESBL-GNB carriers we did not detect rectal colonization. Therefore, samples taken for follow-up of colonization with multi-drug resistant Gram-negative bacteria (MDR-GNB) should ideally also include samples from the site where the MDR-GNB was initially found. Salmonella spp., which suggests that rectal cultures have to be used for follow-up [Infection control guidelines give clear recommendations for the detection of colonization with multidrug-resistant Gram-negative bacteria (MDR-GNB) , 2. RecoIn our tertiary teaching hospital, we have an active screening policy for MDR-GNB, which includes ESBL-producing Gram-negative bacteria. On admission, patients with risk factors for colonization with multidrug-resistant bacteria , are screened for MDR-GNB by rectal culture and when applicable urinary culture (when a urinary catheter is in situ), sputum culture (when the patient is intubated), and wound cultures (in case of open wounds). Patients with known MDR-GNB colonization are nursed with contact precautions and on readmission they are screened by collecting swabs of the rectum and the site where the MDR-GNB was initially found. On the intensive care units and haematology ward selective decontamination of the digestive tract (SDD) is used and active screening cultures are obtained according to standardized protocols: on admission and twice a week thereafter.Pseudomonas and Acinetobacter spp. Criteria for screening for ESBL production were applied according to the Dutch Society for Medical Microbiology guideline (which is largely based on CLSI and EUCAST guidelines): positive ESBL screening was defined as a MIC > 1 mg/L for cefotaxime (or ceftriaxone) and/or ceftazidime [Escherichia coli, Klebsiella spp., Proteus mirabilis, Salmonella spp. and Shigella spp.) confirmation is positive when the zone around the disk/tablet with either cefotaxime or ceftazidime is ≥5 mm larger with clavulanic acid than without; for group 2 Enterobacteriaceae in which chromosomal AmpC beta-lactamases are common  confirmation is positive when the zone around the disk/tablet with cefepime is ≥5 mm larger with clavulanic acid than without [The ESBL-producing Gram-negative bacteria in this study include Enterobacteriaceae and tazidime . PhenotyWe queried our Laboratory Information Management System for all bacterial cultures between January 2012 and August 2016 to identify all patients with one or more cultures positive for ESBL-GNB. All cultures of patients included in the analysis were studied, which include both screening and diagnostic specimen cultures.To assess the value of rectal swab culture we performed a subgroup analysis on patients for whom at least one rectal swab specimen was available. This subgroup was further divided in patients that had been admitted to the ICU or haematology wards (where SDD is used) at least once, and patients that had never been admitted to one of these wards.We identified 1011 patients with one or more cultures positive for ESBL-GNB, which yielded 16,578 specimens for analysis. Per patient a median of 9 samples (inter quartile range 4 – 20) were obtained, the median interval between the first and the last sample obtained was 88 days (interquartile range 8 - 518). The distribution of colonization sites in these patients is shown in Table For 588 patients at least one rectal swab specimen was available . In this subgroup, colonization was detected in the rectal swab culture only in 326 of 588 ESBL-GNB carriers (55.4%). In 82 patients (13.9%) rectal swab cultures remained negative. Colonization was detected both in the rectum and at a different culture site in 180 patients (30.6%). The distribution of colonization sites in these patient groups is shown in Table In this study we detected rectal colonization in 86% of patients colonized with ESBL-GNB and in 14% of ESBL-GNB carriers, no rectal colonization was detected. These 14% of ESBL-GNB carriers might potentially be missed if followed with rectal cultures only. This finding is in line with the scarce literature available on ESBL colonization. Papst et al. found that in 114 patients, 10% of those with positive sample sets had negative rectal swabs ; TschudiOf course, our results are limited by the retrospective nature of the analysis. However, the standardized protocols for ESBL-GNB detection and follow-up at our centre and the relatively large sample size add to the validity of the retrospective analysis. Ideally, rectal samples would have been taken simultaneously with positive ESBL-GNB samples from non-rectal body sites. Therefore we have studied the number of negative rectal swab cultures and interval between an ESBL-GNB positive culture and negative rectal swab culture in the colonized patients without apparent rectal colonization. Thirty-nine percent of patients had negative rectal cultures taken within 8 days of an ESBL-GNB positive sample at another location: loss of colonization within this interval seems not likely. This finding might partly overcome the objection that the absence of rectal colonization in 14% of ESBL-GNB carriers might be caused by sampling bias. In patients with longer intervals between negative rectal samples and ESBL-positive samples from non-rectal sites, natural loss of colonization, the use of antibiotics and/or SDD might have led to negative rectal samples. However, we found similar rectal colonization rates in patients that presumably received SDD (e.g. were admitted to the ICU or haematology ward at some point in the study period), compared to patients that presumably did not receive SDD (e.g. were never admitted to the ICU or haematology ward during the study period). Although this study this has its limitations, we feel that our data can be of help to formulate infection control guidelines. Data on MDR-GNB colonization sites is scarce; more insight in colonization sites is urgently needed, and in the absence of large prospective studies, retrospective analysis with considerable sample sizes are welcomed. Future prospective trials should ideally use standardized sample protocols that will limit the time between rectal sampling and positive ESBL-GNB cultures at non-rectal sites. It is also advisable that such trials would focus on the cost-effectiveness of screening protocols.It is of interest that we found that 28 of 506 patients with positive rectal cultures had a negative rectal sample obtained between two positive rectal samples. This could represent either loss of colonization or sampling error. The latter is the reason why in the Netherlands we require a patient to have two consecutive samples negative for MDR-GNB, before isolation precautions can be discontinued. However, when implementing MDR-GNB screening protocols the potential gain of extra cultures  has to be balanced against the economic impact of performing more cultures. In addition, local and national screening protocols should be adapted to the local and national MDR-GNB epidemiology. In the Netherlands the prevalence of ESBL-producing Enterobacteriaceae is estimated to be around 10% in clinical isolates [In this study we questioned whether our current practice of MDR-GNB follow-up by rectal cultures plus a culture from the site where the MDR-GNB was initially found is more sensitive than follow-up by rectal cultures only. In conclusion, our data and the current available evidence indicate that 10 to 30% of colonized patients do not carry ESBL-GNB in the rectum . This fi


The frontal eye fields (FEFs) and the anterior cingulate cortex (ACC) are commonly coactivated for cognitive saccade tasks, but whether this joined activation indexes coordinated activity underlying successful guidance of sensorimotor mapping is unknown. Here we test whether ACC and FEF circuits coordinate through phase synchronization of local field potential and neural spiking activity in macaque monkeys performing memory-guided and pro- and anti-saccades. We find that FEF and ACC showed prominent synchronization at a 3–9 Hz theta and a 12–30 Hz beta frequency band during the delay and preparation periods with a strong Granger-causal influence from ACC to FEF. The strength of theta- and beta-band coherence between ACC and FEF but not variations in power predict correct task performance. Taken together, the results support a role of ACC in cognitive control of frontoparietal networks and suggest that narrow-band theta and to some extent beta rhythmic activity indexes the coordination of relevant information during periods of enhanced control demands. Frontal eye fields (FEF) and anterior cingulate cortex (ACC) are coactivated during cognitive tasks, but the precise format of their interaction is not known. Here the authors show that phase coupling between ACC -FEF in theta and beta frequency bands better predicts behavioural performance. The frontal eye field (FEF) plays a crucial role in overt135One proposed mechanism for the communication of spatially dispersed neuronal groups is through synchronizing the excitability phases of band-limited activity12121517181920Here we recorded local field potentials (LFPs) and spiking activity from FEF and ACC simultaneously and investigated task-dependent synchronization between the two areas while monkeys performed a memory-guided saccade task and a task with randomly interleaved pro- and anti-saccade trials. The results show that a narrow-band theta and beta rhythmic activity indexes the coordination of relevant information between FEF and ACC during periods of enhanced control demands.n=233 sites; n=87 and n=146 in monkey 1 and 2, respectively) and FEF  while monkeys performed the memory-guided saccade task and the pro-/anti-saccade task  and FEF units . Both ACC and FEF LFPs showed a prominent increase in theta power (3–9 Hz) during the delay period . AdditioP<0.05, Binomial test) but not ACC  channels showed significant spatial tuning of power within the theta frequency band (3–9 Hz) during the delay period in the time window of 400–1,100 ms following target stimulus onset (P<0.05). In addition, a significant number of FEF  but not ACC channels  showed significant tuning in the beta band (12–30 Hz). Thus, we found spatial tuning of LFP power in theta and beta frequencies in FEF but not in ACC. The observed power modulation of the FEF LFPs is in line with previous FEF single-unit recording results, which suggest a high degree of spatial tuning in FEF neuronsWe further examined whether LFP power was tuned to the spatial target location. We found that a significant number of FEF . On the other hand, respectively, 128 (18.99%) and 179 (26.56%) pairs exhibited statistically significant decreased theta and beta synchrony in the delay period relative to the baseline . We observed that, respectively, 234/674 (34.72%), 213/674 (31.6%), and 96/674 (14.83%) of channel pairs exhibited increased phase synchronization across both theta and beta band, theta band only, and beta band only, during the delay period versus baseline. With regard to significant decreased phase synchrony in the delay period versus baseline, the following results were obtained: 39/674 (5.79%) channel pairs exhibited concurrent theta- and beta-band decrease, 89/674 (13.2%) pairs displayed theta- but not beta-band decrease, and 140/674 (20.77%) showed beta- but not theta-band phase synchrony decrease. Furthermore, the population of ACC-FEF channel pairs exhibited increased theta- and beta-band synchrony in the delay period versus baseline of the memory-guided saccade task (We next tested whether ACC and FEF showed task-dependent interactions in the theta (3–9 Hz) and beta (12–30 Hz) band. For a large subset of interareal recording pairs, theta- and beta-band phase synchronization increased from the baseline  to the delay period of the memory-guided saccade task . Of the 674 ACC-FEF LFP-LFP channel pairs, respectively 447 (66.32%) and 330 (48.96%) channel pairs displayed significant increases in theta- and beta-band phase synchronization during the delay period , as shown in We further investigated the directionality of the ACC-FEF interactions by performing Granger-causality analyses. Both during the delay and the baseline period, we observed bidirectional Granger-causal influences between ACC and FEF LFPs, with prominent peaks in the theta and beta range, and a significantly stronger influence from ACC to FEF than vice versa . We validated the relationship of coherence with performance by using an anti-saccade task that triggered more directional error trials than the memory-guided saccade task. Similar to the memory-guided saccade task, the population of the ACC-FEF channel pairs showed significantly decreased theta- and beta-band phase synchronization in the error versus correct anti-saccades during the 400–1,100 ms following the fixation onset during the preparatory period . We confirmed that these statistical differences were not the result of differences in sample size, by performing the same analysis using the same number of trials for correct and error trials.We found that FEF and ACC LFPs exhibit phase synchronization in the theta and beta frequency bands during the delay period. Our next question was whether this synchronization predicted behavioural task performance. To test whether the strength of phase synchronization influenced performance in the memory-guided saccade task, we compared FEF-ACC phase synchronization between correct trials and error trials in which the animal made an eye movement toward the wrong stimulus location. We found that both theta- and beta-band coherence between the ACC and FEF were significantly larger on correct trials than error trials during the delay period  as a measure of spike-field coherenceP<0.001; FEF units: 16/154=10.4%, P<0.001, Binomial test) and ipsiversive trials . In contrast, we found no evidence for significant decreases in phase locking  and ipsiversive trials , whereas we found no evidence for significant decreases   in the delay period versus baseline of both contraversive   right. Mal test) , right. al test) .We also tested for possible influences of firing rate differences between units on the observed spike LFP synchronization. We repeated the spike-field analysis, but only including in the analysis those units with no significant difference of spike rates between the tested conditions. The obtained effects were still statistically significant  was evident in more than half of the LFP-LFP recording pairs, (2) translated to spiking activity in the ACC and FEF with significant interareal spike-LFP synchronization in both anatomical directions, that is, with spikes from ACC coupled to LFPs in FEF and with FEF spikes coupling to LFPs from ACC, (3) indexed correct versus erroneous working memory performance, and (4) exhibited asymmetric directionality, with stronger influence of ACC over FEF than vice versa. These findings provide evidence that functional interactions between brain areas implementing (associated with FEF) and biasing (associated with ACC) working memory and also higher-order cognitive performance proceed through phase-synchronized activation at band-limited theta and beta rhythmic activity1516Neuronal circuits in FEF are well known to encode target locations for overt orienting and covert stimulus selection during working memory, sustained attention, and visual search tasks 14We found that a moderate fraction of ACC and FEF units synchronized to theta LFP activity at distant sites  more strongly during the working memory delay than during the predelay baseline period see  and 6. T153941The role of frontal midline theta in working memory has been previously demonstrated in human studies and the ACC has been suggested as its main sourceAs mentioned in the introduction, beta-band synchronization is suggested to be involved in long-range transmission of information between brain areas171819142027One possible mechanism for ACC-FEF phase synchronization could be based on interactions of a subclass of parvalbumin expressing, fast spiking interneurons, and subsets of pyramidal cells showing resonance to theta rhythmic inhibition14We observed that the theta-band Granger causality increased in the delay period in both ACC→FEF and also FEF→ACC directions. This could indicate that theta-band synchrony is involved in both top-down and bottom-up processes, as has been described previously15In summary, the increased ACC-FEF phase synchrony and also increased influence of the ACC over FEF during the delay period suggests that the ACC-FEF interaction serves a functional role. The increased ACC unit-FEF LFP coupling in the delay period of the contraversive saccades could indicate that the ACC biases FEF to generate a contraversive saccade . Our res1431Macaca mulatta and Macaca fascicularis) weighing 7–9 kg were subjects in this study. Recording chambers were implanted over the right arcuate sulcus and right anterior cingulate sulcus based on previously obtained MRIs. Details for the surgical procedures have been described previouslyTwo male adult macaque monkeys  the colour of which instructed the monkeys to perform a pro- or anti-saccade on stimulus presentation. The fixation point remained illuminated for a period of 1,100–1,400 ms, which we designate as the preparatory period. The monkeys were required to maintain fixation during the preparatory period and then the target stimulus appeared at a distance of 9° either to the right or left of the central fixation point. In pro-saccade trials, the monkeys were required to saccade towards the target stimulus, and in anti-saccade trials, they had to make a saccade towards the mirror location of the target stimulus . To obtaet al.We simultaneously recorded the LFP and unit activity in the ACC and FEF while monkeys performed a memory-guided saccade task and a randomly interleaved pro-/anti-saccade task . FEF andThe signal from each electrode was preamplified (Plexon Inc.) and band-pass filtered between 0.5–250 Hz for LFP signals and between 250 and 8,000 Hz for the single /multiunits and digitized at the frequency of 40 kHz. Each area's signal was referenced separately against the corresponding recording chamber. The sampling rate for LFP signals was set at 1,000 Hz. ACC and FEF multineuron activity was isolated offline by applying a threshold±3–5 s.d. above or below the average raw signal to separate the multineuron activity from noise. We further visually inspected the waveforms to confirm that this procedure resulted in normal waveforms and the multiunits with abnormal waveform shape were excluded from the analysis. We will refer to multineuron activity as units throughout the paper.http://www.ru.nl/fcdonders/fieldtrip/)z-score normalized using a baseline of 500–1,000 ms prior to the fixation point onset in both memory-guided saccade task and pro-/anti-saccade task.All of the data analyses were performed with custom-written Matlab code  using the fieldtrip toolbox , we considered the difference between groups as significant. We obtained statistically significant results in all the comparisons that t-test yielded statistical significance; thus, we could validate the t-test results with this permutation test.Here The WPLI-debiased method outputs a single value for a given trial set and thus, we cannot perform a traditional statistics to test whether there is a difference in WPLI-debiased coherence between the baseline and delay period of a single channel pair. To test for a statistically significant difference in coherence between baseline (700 ms before fixation onset) and delay  period in a single channel pair, we used a bootstrapping approach. We created 1,000 bootstraps with replacement of the original trial set and obtained a distribution of the WPLI-debiased for the baseline and delay periods. The difference between baseline and delay was determined to be significant if the mean of the WPLI-debiased in the delay period fell above or below the 99.9th percentile of the WPLI-debiased distribution of the baseline period. To further confirm the results of the bootstrapping procedure, we pooled the baseline and delay WPLI-debiased results and randomly assigned them into two groups. Then, we tested whether the mean difference between the original baseline and delay period falls above or below the 99.9th percentile of the mean difference of the randomly generated groups. The results of this procedure yielded nearly identical results to the bootstrapping procedure we have described above.et al.We have subtracted the evoked response from the raw data to investigate whether or not the observed phase synchronization is due to the induced oscillations. To further examine whether the observed synchronization was due to the induced oscillations, we have subtracted the evoked response using a method described by Truccolo To analyse the directionality of interactions, we performed Granger-causality analysis. Granger-causality analysis is based on modelling the two time series as a vector autoregressive model,M=32 and using the armorf function in the BSMART toolboxet al.In this equation, the two signals are predicted by a linear combination of their own past values, and the past values of the other time series. The residual errors (unexplained variance) are captured by the variables x2 to x1 is now defined asThe frequency-domain Granger causality from f over the amount of power that remains after the predictions from the second signal have been factored in.This expression can be understood as the log fraction of intrinsic power in the first signal at frequency x1 to x2 is stronger than the Granger causality from x2 to x1, then we expect that the reverse holds true when we time reverse the signalset al.It is well established that Granger causality can be sensitive to both uncorrelated and correlated noise that is superimposed on the measurements of the signals of interest. This can lead to a substantial amount of false alarms in the identification of Granger-causality relationships see refs , 67, 68.t-test. We further confirmed the results of the t-test by implementing a permutation test. The permutation test procedure is described in detail above.The Granger-causal influence of the ACC over FEF is shown as GACC→FEF and the Granger-causal influence of FEF over ACC is denoted as GFEF→ACC. The overall influence of ACC over FEF was derived by implementing the (GACC→FEF−GFEF→ACC) equation. The results shown in We also shuffled the time-frequency transformed raw data and computed the corresponding Granger values to obtain the chance level Granger. Then we compared the chance level GACC→FEF and GFEF→ACC values to the original Granger values from the non-shuffled data set. We observed that across theta and beta frequencies, both the GACC→FEF and GFEF→ACC values in the baseline and delay periods were above the chance level.We used the PPC as a measure of spike-field synchrony; for a detailed mathematical definition see ref. M is the number of spikes, Ui is the vector describing the x and y component of the spike-LFP phase for the ith. We only included units with at least 50 spiking events in the tested conditions to obtain a reliable and robust results and increase the statistical power of the analysis as has been shown before15t-test and also a permutation test to probe the significant difference of the PPC across experimental conditions. We have described the permutation procedure above. To correct for multiple comparisons, we have implemented the false discovery rate algorithm as described by Benjamini and YekutieliHere To test for significant spike-field coupling across single ACC-unit with FEF-LFP and FEF-unit with ACC-LFP, we performed a permutation test. We first randomly shuffled the spikes across the tested conditions  and obtained a distribution of the PPC values in the randomized experimental conditions. Then, we tested whether the PPC values of the original non-randomized conditions fell above or below the 2.5th percentile of the randomization distribution. If so, we designated that unit-LFP pair as having a significant modulation across the tested conditions.We have not made the raw data available. However, the computer codes used to perform data analysis could be provided on request.How to cite this article: Babapoor-Farrokhran, S. et al. Theta and beta synchrony coordinate frontal eye fields and anterior cingulate cortex during sensorimotor mapping. Nat. Commun.8, 13967 doi: 10.1038/ncomms13967 (2017).Supplementary Figures and Supplementary References


DNA methylation is the most characterized epigenetic process exhibiting stochastic variation across different tissues and individuals. In non-invasive prenatal testing (NIPT) fetal specific methylated regions can potentially be used as biomarkers for the accurate detection of fetal aneuploidies. The aim of this study was the investigation of inter-individual methylation variability of previously reported fetal-specific markers and their implementation towards the development of a novel NIPT assay for the detection of trisomies 13, 18, and 21. Methylated DNA Immunoprecipitation (MeDIP) combined with in-solution targeted enrichment followed by NGS was performed in 29 CVS and 27 female plasma samples to assess inter-individual methylation variability of 331 fetal-specific differentially methylated regions (DMRs). The same approach was implemented for the NIPT of trisomies 13, 18 and 21 using spiked-in (n = 6) and pregnancy samples (n = 44), including one trisomy 13, one trisomy 18 and four trisomy 21. Despite the variability of DMRs, CVS samples showed statistically significant hypermethylation (p<2e-16) compared to plasma samples. Importantly, our assay correctly classified all euploid and aneuploid cases without any false positive results (n = 44). This work provides the starting point for the development of a NIPT assay based on a robust set of fetal specific biomarkers for the detection of fetal aneuploidies. Furthermore, the assay’s targeted nature significantly reduces the analysis cost per sample while providing high read depth at regions of interest increasing significantly its accuracy. The current gold standard in prenatal diagnosis involves invasive testing of fetal DNA through amniocentesis and chorionic villus sampling (CVS). These procedures are associated with a considerable risk of spontaneous abortion, estimated at 0.1–0.2% .In the last decade several approaches have been developed for NIPT of fetal aneuploidies, including DNA-based approaches, investigation of targeted-fetal specific mRNAs  or fetalThe advent of next generation sequencing (NGS) has greatly facilitated the development of NIPT. Initial efforts using massive parallel sequencing (MPS) showed high potential in the non-invasive detection of fetal aneuploidies ,9. More Using MeDIP coupled with high resolution array-CGH, our group identified more than 2000 DMRs on chromosomes 21, 18, 13, X and Y . AdditioThe study was approved by the Cyprus National Bioethics Committee and informed written consent was obtained from all participants. For the investigation and characterization of inter-individual methylation variability of selected DMRs, 29 first trimester (11–14 weeks of gestation) CVS (24 euploid and five trisomy 21) and 27 female non-pregnant plasma samples were used. All CVS underwent karyotyping and/or Quantitative-Fluorescent PCR (QF-PCR) analysis to confirm their status. Non-pregnant female plasma samples were obtained from Sera Laboratories International Ltd .For the development of the NIPT assay, 44 peripheral blood samples were collected into two 8 mL EDTA-containing tubes from women with singleton pregnancies (11–14 weeks of gestation). A mean of 8 ml plasma was isolated via a double centrifugation protocol, as previously described . All preGenomic DNA was extracted from CVS samples using the QIAamp Mini kit  according to the manufacturer’s instructions. Cell-free DNA was extracted using the QIAsymphony DSP virus/pathogen midi kit (Qiagen) on QIAsymphony SP/AS.Total cfDNA concentration and fetal fraction were estimated using Taqman probes, targeting the DYS14 and β-globin loci as previously described , or an iTowards the development of a NIPT assay, our initial efforts were focused on the implementation of MeDIP combined with in-solution targeted enrichment on synthetic affected and non-affected plasma samples. DNA obtained from plasma of non-pregnant female samples were spiked-in with DNA obtained from euploid or trisomy 21 male CVS samples at concentrations of 5%, 10% and 20%. Before mixing, fetal DNA was sheared to an average size of 230bp using the Bioruptor Twin Sonicator . The concentration of spiked-in DNA into plasma DNA was measured using qPCR using the DYS14 and β-globin loci as previously described [DNA from CVS and non-pregnant female plasma were used to generate libraries using standard preparation methods. Genomic DNA obtained from CVS samples ranging from 12–30 ng was sheared to an average size of 230bp using the Bioruptor Twin Sonicator  and run on the TapeStation 2200  for fragment size verification. Blunt-ending and sequencing-adaptor ligation were performed prior to MeDIP using NEB Blunting and Ligase enzymes  as previously described ,24,25. FTarget capture probes 140-160bp) were designed to enrich selected fetal-specific DMRs across different chromosomes, as previously described 0bp were . CaptureTarget capture probes were used to enrich libraries using in-solution Hybridization ,28. EachAdaptor sequences were trimmed with cutadapt v.1.2 and sequencing reads were aligned to the human reference genome GRCh37/hg19 (NCBI build 37) using the BWA v.0.7.4 MEM algorithm . FollowiIn order to assess the inter-individual methylation variability of selected DMRs in CVS and non-pregnant female plasma samples, we calculated their respective standard deviation and standard error for each DMR using their normalized read depth. Normalization was performed by equalizing the cumulative read depth of all DMRs across all samples. The 95% confidence intervals that were calculated provided information about the variability of each DMR. Additionally, by comparing their confidence intervals, the discrimination of CVS and non-pregnant female plasma samples methylation levels was derived for a given DMR. Potential overlap between the intervals suggests that there is no significant methylation difference between CVS and non-pregnant female plasma samples whereas lack of overlap suggests the possibility for discriminating the two tissues based on their methylation level.For the classification of pregnancies, a LOESS normalization model  was initially employed to alleviate GC-bias. The model was fitted only on regions that were not located on potentially trisomic chromosomes  and the model’s predicted values were obtained for all regions. The normalized read depths were taken to be the ratio of the observed to the predicted read depths. Following the GC-normalization, the resulting normalized read depths were used in a two-sample t-test, where the DMRs of the potentially trisomic chromosome were compared to the remaining DMRs. The z-scores were calculated as a Welch's t-statistic test intended for use with two samples with potentially unequal variances.Inter-individual methylation variability of the previously confirmed 331 fetal-specific DMRs  was ascePairwise methylation comparisons for each DMR between CVS and female plasma samples indicated that out of the 331 DMRs, 313 showed significant hypermethylation in CVS compared to plasma samples  .The methylation enrichment in CVS was on average two fold higher compared to female plasma samples with mean enrichment values  of 108 and 56 respectively . The coeIn order to identify DMRs that enable the best discrimination between fetal and maternal DNA, we first proceeded by adjusting the test’s p-values using the Tukey’s HSD method. A threshold of 0.05 was chosen for the adjusted p-values for differences between CVS and non-pregnant female plasma samples. In total 78 DMRs successfully passed the specific threshold . For thiTowards the development of a NIPT assay for the detection of fetal aneuploidies, our initial efforts were focused on the implementation of MeDIP combined with in-solution targeted enrichment followed by NGS using three euploid and three trisomy 21 spiked-in samples simulating 5%, 10% and 20% fetal fractions. For the classification test, the read depth of each DMR was first normalized using a LOESS model on read depth against GC content in order to remove potential GC-bias in sequencing data. A z-test was then applied to each sample that compared the normalized read depth of DMRs on chr21 (region of interest) against the normalized read depth of DMRs on the other autosomes (reference). The median z-score of the three euploid spiked-in samples was subtracted from all six z-scores in order to center the euploid samples around the value of zero. Since a scale normalization was not appropriate with six samples, we did not use a hard threshold for classification, instead, this study assessed our ability to distinguish aneuploid samples in a series of dilutions. The z-scores of the six spiked-in samples showed a clear discrimination between the scores of the euploid and trisomy 21 samples .In addition, there was a clear trend in the scores’ increase with respect to the fetal fraction of the aneuploid samples. Euploid samples remained unaffected by the increased fetal fraction.Following the assessment of the NIPT assay on spiked-in samples, we tested its performance in a proof-of-concept study for the detection of fetal aneuploidies  in real pregnancy plasma samples. The study included 38 euploid and six aneuploid plasma samples  obtained from women with singleton pregnancies (11–14 weeks of gestation). The observed fetal DNA fraction ranged from 5% to 16% with a median value of 11% .GC-bias correction with LOESS and z-score calculation was performed as described in the Data Analysis section. Further score normalization was carried out by subtracting the median score value from the euploid samples, as described in the synthetic pregnancy samples. In addition, scale normalization was performed by dividing all z-scores by twice the standard deviation of the scores from the euploid samples. Therefore, a discriminating threshold of two standard deviations away from the median translated to a threshold value of 1. All four trisomy 21 as well as the trisomy 18 and trisomy 13 cases were correctly classified . No false positive samples were detected .An optimal panel of fetal-specific DMRs for NIPT should eliminate maternal background and show high homogeneity between individual fetuses . Based oOverall methylation analysis showed that despite the inter-individual methylation variability, the tested DMRs showed differential enrichment between fetal DNA (CVS) and maternal DNA . Specifically, CVS samples showed statistically significant hypermethylation (p<2e-16) as compared to plasma samples with mean enrichment values of 108 and 56 respectively. Additional statistical analysis of the methylation status of the initial set of 331 DMRs revealed that 78 DMRs  showed the best discrimination between fetal and maternal tissues. Based on our statistical classification model this number is not sufficient for clinical NIPT applications, especially considering the limited number of DMRs in the potentially aneuploid regions . Even though a small subset of potential DMRs exhibited p-values higher than our specified threshold (p = 0.001) we still included them in our assay since even for these markers the extra copy of a potential trisomic sample can still contribute towards the classification power of the test.Additionally, we developed and assessed the performance of a novel NIPT assay for the detection of the most common fetal aneuploidies during pregnancy . For the first time, we implemented MeDIP in combination with in-solution hybridization on spiked-in and pregnancy plasma samples which resulted to the successful classification of all euploid and aneuploid samples. Specifically, implementation of our double enrichment approach on spiked-in samples detected all trisomy 21 spiked-in samples as they showed higher z-scores values than the respective z-score values of euploid spiked-in samples. Subsequently, the performance of this novel method was evaluated on pregnancy cases for aneuploidy detection. This proof-of-concept study provided an accurate detection of all trisomy 21 (n = 4), trisomy 18 (n = 1) and trisomy 13 (n = 1) samples using a total of 44 pregnancy samples whereas no false positive samples were observed. This novel assay provides the starting point for the development of an alternative and cost-effective non-invasive method for the detection of fetal aneuploidy from maternal plasma. Our approach increases the diagnostic accuracy as it enables a very high read depth and robust analysis of the targeted DMRs. In addition, the targeted enrichment of multiple DMRs reduces the sequencing cost per sample as it enables a higher number of samples to be analyzed simultaneously.The classification of fetal trisomies was performed using 98 markers  . Future Overall, the method described in this study provides unbiased and accurate results for a wide range of cfDNA concentrations at different fetal fractions, ranging from 5–16%. In our proof-of-concept study we have correctly classified all euploid and aneuploid cases. Previous studies have utilized a large number of samples for validation purposes ,43. ThusInvestigation of inter-individual methylation variability on selected DMRs showed that despite the presence of variability, there is a distinct and robust difference between fetal and maternal tissues. In addition, we are reporting for the first time the development of a novel assay for the detection of fetal aneuploidies from maternal plasma samples, using MeDIP in combination with in-solution targeted enrichment followed by NGS. In a proof-of-concept study presented herein, our assay correctly classified all euploid and aneuploid cases without any false positive results (n = 44). However, a larger validation study including more aneuploid samples and more DMRs should be performed to further assess the diagnostic sensitivity and specificity of the assay. In addition, the assay’s targeted nature significantly reduces the analysis cost per sample while providing high read depth at regions of interest increasing significantly its accuracy.Conclusively, our work provides the starting point for the development of a clinical NIPT assay for the detection of fetal aneuploidies using a robust and well-characterized set of fetal specific biomarkers and provides the possibility for the expansion of the existing disease panel to include detection of microdeletion syndromes and potentially monogenic diseases.S1 FigSquares show the mean methylation enrichment for the CVS (blue) and non-pregnant female plasma samples (orange). The extended lines show the distance of one standard error (plus and minus). All DMRs exhibited distinct and consistently higher methylation levels among the 29 CVS samples as compared to the 27 non-pregnant female plasma samples. The 78 markers were chosen so their Tukey HSD adjusted p-values are less than 0.05.(TIFF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.