Controlling the nature of the electronic states within organic layers holds the promise of truly molecular electronics. To achieve that we, here, develop a modular concept for a versatile tuning of electronic properties in organic monolayers and their interfaces. The suggested strategy relies on directly exploiting collective electrostatic effects, which emerge naturally in an ensemble of polar molecules. By means of quantum‐mechanical modeling we show that in this way monolayer‐based quantum‐cascades and quantum‐well structures can be realized, which allow a precise control of the local electronic structure and the localization of electronic states. Extending that concept, we furthermore discuss strategies for activating spin sensitivity in specific regions of an organic monolayer. In many instances, a convenient way for tuning the characteristics of such interfaces is by using molecular (mono)layers. These can even adopt the role of functional elements in devices, for example, in highly efficient organic monolayer transistorsIn “classical” quantum‐devices based on inorganic semiconductors, such as quantum‐cascade lasers,FigureIn the present contribution we suggest a conceptually different approach, developing the concept of a powerful and versatile toolbox that consists of functional elements for the realization of organic SAMs in which the energetics and orbital localization can be efficiently tuned. As will be shown, well‐known electrostatic effects can exploited to design SAMs with unprecedented electronic properties. As an exemplary case for our discussion we chose conjugated thiols that can be covalently bonded to Au surfaces , see Figuren = 2) has been chosen, as pyrimidine‐containing systems have large dipole moments,For the molecular backbones discussed in the following examples, we picked the first structure contained in Figure The above choice leads to the structure shown in Figure FigureEF) and the lowest unoccupied states on the right one (0.6 eV above EF). This strong localization is also well visible in the isodensity plots of the LDOS at the specific energies corresponding to the frontier states . An equivalent situation is found for the right semiconducting segment. The change in the electrostatic environment of each segment of the monolayer and the associated shifts and localization of electronic states directly arises from the collective interaction of the embedded polar segments within the monolayer. Hence, it is not surprising that in the limit of very low coverage as well as for the isolated molecule, where collective effects vanish, the asymmetry between the left and right semiconducting units are reduced by a factor of ≈2.6 .Beyond controlling the energies of the electronic levels, the localization of the relevant frontier states is another crucial aspect in the design of interfacial quantum‐structures. We visualize the localization of the electronic states using a local density of states (LDOS) contour map, which displays the charge density as a function of energy  and position along the molecular backbone , even quantum‐cascades can be realized in the spirit of Figure 2.4Following the general layout of the quantum‐cascade structure, but flipping the orientation of one of the involved dipolar segments, the quantum‐well like structures presaged in Figure 2.5FigureTo realize a locally spin‐polarized monolayer the next element of the toolbox proposed in Figure EF. This local spin‐polarization is also clearly visible in the LDOS contour maps in Figure The spin‐polarized nature of the electronic states in the B‐substituted pyrene segment (green) is clearly visible in the density of states projected onto the individual segments of the SAM shown in Figure 2.6As an appealing outlook for future efforts, the development of linker elements stabilizing SAMs  is proposed. Designing the necessary molecules represents a clearly more involved task, as it inherently requires a spatial extent of functional elements that is compatible with inter‐molecular distances to be able to form inter‐molecular bonds. The inter‐molecular distances are in turn determined by the immediate substrate–organic interface . Thus, while H‐bonded SAMs have been discussed in a few instances in the literature,3The above‐discussed examples show how the electronic structure of a SAM can be manipulated through collective electrostatic effects arising from the parallel alignment of dipolar building blocks. Going substantially beyond previous work, we have shown that combining them with semiconducting elements allows for the realization of quantum‐cascades or monolayer‐based quantum‐well structures. Furthermore, by including neutral radical segments into the SAMs, even locally spin‐polarized monolayers can be realized. While challenging to realize experimentally, these examples only serve as a first showcase hinting towards the potential of such an “electrostatic” toolbox for manipulating electronic properties of interfacial layers, which could be easily expanded by varying the band‐gaps of the semiconducting elements and the magnitudes of the potential shifts due to the dipolar units. Also alternative functional elements can be envisioned, such as chemical linkers or electronically decoupling sections. Thus, our results only mark the first step towards the development of a highly versatile and flexible toolbox.4Computational Approach: All calculations discussed in this paper have been performed in the framework of DFT. The displayed results have been obtained using periodic boundary conditions and the PBE−3 eV−1). The density of states was integrated in an energy window with a width of 0.1 eV around the peak positions.System Setup: The monolayer structures were obtained using the following approach: Molecular geometries were first optimized using Gaussian03As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


P = 0.003, OR = 0.68 (0.52–0.88)).Sarcoidosis is a multisystem granulomatous disease of unknown aetiology characterized by increased inflammation, and results from gene–environment interactions. Proteinase‐activated receptor‐1 mediates the interplay between coagulation and inflammation. The rs2227744G > A promoter single nucleotide polymorphism has been linked to inflammation, cardiovascular disease and chronic obstructive pulmonary disease exacerbations. Using a case‐control study (184 cases with sarcoidosis and 368 controls), we show that the rs2227744A allele significantly associates with protection from sarcoidosis ( CIconfidence intervalCOPDchronic obstructive pulmonary diseaseELSAEnglish longitudinal study of ageingF2Rfactor 2 receptor1FEVforced expiratory volume in 1 sFVCforced vital capacityHLAhuman leukocyte antigenHWEHardy–Weinberg equilibriumMHCmajor histocompatibility complexORodds ratioPAR‐1proteinase‐activated receptor‐1SNPsingle nucleotide polymorphismSarcoidosis is a multisystem granulomatous disease of unknown aetiology.F2R) gene (chromosome 5q13.3) has been demonstrated to confer higher promoter activity, and hence higher PAR‐1 expression levels, in vitro.Activation of proteinase‐activated receptor‐1 (PAR‐1) by coagulation proteinases contributes to lung inflammation and fibrosis, and we have shown that PAR‐1 knock‐out mice are protected when these conditions are experimentally induced.A UK Caucasian White population of 184 sarcoidosis subjects recruited from University College London Hospital, the Royal Free Hospital and St George's Hospital was used for this study.Genotyping of the Whitehall II and ELSA cohorts was performed by KBiosciences using the KASPar assay , based on Kompetitive Allele Specific PCR or KASP technology. For the sarcoidosis cohort and National Blood Service samples, a region containing the rs2227744G > A polymorphism was PCR amplified and the products sequenced using the MegaBACE automated sequencing and a MegaBACE 1000 DNA analysis system  following the manufacturer instructions. Samples were analysed using a MegaBACE 1000 DNA analysis system.t‐test. For genotypic data, all procedures were performed using PLINK v1.07.For phenotypic data, categorical data were tested for differences between cases with persistent and non‐persistent disease by means of a chi‐square test, and continuous data were tested using the 2‐tailed Student 1/FVC) ratio of 70% or greater as would be expected.The characteristics of the sarcoidosis patients and healthy controls are reported in Table A large cohort of 8579 UK Caucasian healthy individuals, who participated in the ELSA or Whitehall II study, was previously genotyped for this polymorphism.P‐value = 0.62) or controls  or both . Association analyses showed that the rs2227744A minor allele is associated with protection from sarcoidosis but not with persistent and non‐persistent phenotype. Allelic chi‐square analyses of cases and controls showed that the rs2227744A allele was significantly less frequent in cases compared with controls, with the additive model being the most significant ). The association was confirmed by genotypic analyses   or genotypic level   who collected the ELSA samples used in this study and all the participants. We thank Dr Huw Beynon and Dr Paul Dilworth , Dr Charlotte Rayner  and Kevin Hart  for providing access to sarcoidosis patients and controls. We are also very grateful to Cathy Read who collected blood samples at the UCL Hospitals. We would finally like to thank all the patients participating in the Sarcoidosis cohort. We also wish to thank Dr Ilaria Guella (UBC) for helpful discussion on data analysis. This work was supported by a British Lung Foundation, Garfield Weston Foundation award (Grant reference COPD10‐6), a Wellcome Trust Clinical Training Fellowship (074558) and a Wellcome Trust program grant (051154). The Whitehall II study has been supported by grants from the Medical Research Council (MRC); Economic and Social Research Council; British Heart Foundation ; Health and Safety Executive; Department of Health; National Heart Lung and Blood Institute (HL36310); US NIH National Institute on Aging (AG13196); US NIH Agency for Health Care Policy Research (HS06516); and the John D. and Catherine T. MacArthur Foundation Research Networks on Successful Midlife Development and Socioeconomic Status and Health. The US National Institute on Aging  funds the English Longitudinal Study of Ageing DNA Repository (EDNAR), and ELSA is funded by the National Institute of Aging in the USA and a consortium of UK Government departments coordinated by the Office for National Statistics. We declare that the funders of this project played no role in the design of the study, the collection and analysis of the data, or in the preparation of the manuscript.


To maximise the potential benefits of maternity care services, pregnant women need to be able to physically get to health facilities in a timely manner. In most of sub–Saharan Africa, transport represents a major practical barrier. Here we evaluate the extent to which an innovative national ambulance service in Ethiopia, together with mobile phones, may have been successful in averting pregnancy–related deaths.An operational assessment of pregnancy–related deaths in relation to utilisation of the new national ambulance service was undertaken in six randomly selected Districts in northern Ethiopia. All 183 286 households in the six randomly selected Districts were visited to identify live–births and deaths among women of reproductive age that occurred over a one–year period. The uptake of the new ambulance transport service for women’s deliveries in the same six randomly selected Districts over the same period was determined retrospectively from ambulance log books. Pregnancy–related deaths as determined by the World Health Organization (WHO 2012) verbal autopsy tool [13] and the InterVA–4 model [14] were analysed against ambulance utilisation by District, month, local area, distance from health facility and mobile network coverage.P = 0.01). Distance to a health facility, mobile network availability and ambulance utilisation were all significantly associated with pregnancy–related mortality on a bivariable basis. On a multivariable basis, ambulance non–utilisation uniquely persisted as a significant determinant of mortality .A total of 51 pregnancy–related deaths and 19 179 live–births were documented. Pregnancy–related mortality for Districts with above average ambulance utilisation was 149 per 100 000 live–births , compared with 350 per 100 000 (95% CI 249–479) for below average utilisation (The uptake of freely available transport in connection with women’s obstetric needs correlated with substantially reduced pregnancy–related mortality in this operational assessment, though the design did not allow cause and effect to be attributed. However, the halving of pregnancy–related mortality associated with ambulance uptake in the sampled Districts suggests that the provision of transport to delivery facilities in Africa may be a key innovation for delivering maternal health care, which requires wider consideration. Despite major international concerns around maternal health and institutional delivery rates , little The principle that effective transport for obstetric health emergencies is essential is not a matter for debate in most settings. However, that thinking has not translated widely into sub–Saharan Africa, where access to obstetric care remains a major barrier, with a lack of transportation and other infrastructure. Only a few sub–Saharan countries have considered and evaluated the provision of ambulances to facilitate access to obstetric care even in emergencies, for example in Burundi , Uganda In Ethiopia, the 2011 DHS report found 9.9% of births nationally during the previous five years were delivered at a health facility (10.6% in Tigray Region) and 71.1% of women mentioned lack of transport to a facility as a major barrier (52.4% in Tigray Region) [A total of 1250 ambulances have been distributed, with at least one ambulance per District and nearly half of Districts (larger ones) getting two. The total investment to achieve this was about US $50 million. Before ambulances were deployed, the Ethiopian Federal Ministry of Health signed an agreement with governments of the nine regional states and the two autonomous city administrations (Addis Ababa and Dire Dawa) to regulate use. The agreement entailed three important commitments. First the regional governments committed to allocate budgets to cover the running costs for the ambulances; second they committed to replacing the ambulances after five years; and third they agreed to make ambulance services available free of charge. Furthermore, a number of town–hall and community meetings were held to inform communities about these commitments. Once the ambulances were delivered, frequent reminders about the commitments were sent out through local mass media. Laws to enact the ambulance service provision and ensure and safeguard the proper utilisation of ambulances have also been passed by the regional cabinets.tabia) level to data on actual ambulance utilisation for obstetric care, as a means of assessing the extent to which ambulance utilisation may have averted pregnancy–related deaths.These developments in Ethiopia therefore provided a unique opportunity to contribute to filling the current evidence gap on the provision of non–emergency obstetric transport in Africa. An operational assessment of the effectiveness of the innovative ambulance service for transporting women to facilities and averting pregnancy–related deaths was conducted, based on two interlinked data sources. Tigray Regional Health Bureau, in the north of Ethiopia, had previously undertaken a one–year representative randomised population survey of pregnancy–related mortality , which cworeda) in Tigray Region form six geographic Zones, and one District per Zone was randomly selected as a stratified sample, covering a population of 843 115. A two–stage retrospective household mortality survey was carried out in mid–2013 by community health staff in the selected Districts, following up deaths among women of reproductive age (15–49 years) over a one–year period  using the WHO 2012 verbal autopsy tool [The detailed methodology for the pregnancy–related mortality survey design has been presented elsewhere . Brieflypsy tool . The dat age 15–4 years ovAs a separate exercise, the vehicle log books for the ambulances in the six randomly selected districts underwent retrospective data capture for the same one–year period. The data for each trip included whether it was connected with a delivery; from which community it originated; patient’s name; distance travelled; destination and date. The completeness of the vehicle logs was verified using the odometer readings for the start and end of every journey in each vehicle, before the journeys specifically relating to women delivering were extracted.Descriptive statistics were used to characterise the patterns of ambulance transportation in relation to pregnancy–related deaths and determinants including District, month, local area, distance from health facility and mobile telephone coverage. Multivariable Poisson regression modelling was used to assess competing contributions of distance to facility, mobile network availability and ambulance utilisation on pregnancy–related mortality at local area level .During the one–year period, 51 pregnancy–related deaths and 19 179 live–births were identified in the community survey of 183 286 households in the six study Districts, as previously described . CorrespP = 0.01.The proportion of deliveries using an ambulance was calculated for each month and District as a means of tracking patterns in ambulance utilisation over the one–year period. This is shown in P = 0.006) and in the local areas covered by mobile telephone networks .P = 0.03), while distance to the District Health Centre lost most of its effect.A Poisson regression model was constructed including all 131 local areas in the survey, with the number of pregnancy–related deaths as the dependent variable and the number of live–births as the exposure term for each area. Independent variables were distance from the District Health Centre, availability of the mobile telephone network, and ambulance utilisation. By considering These results clearly show substantially lower pregnancy–related mortality in places and periods where free ambulance transport was used by women in connection with their deliveries. We entirely accept that an operational assessment of this kind cannot demonstrate statistically that ambulance utilisation caused reductions in pregnancy–related mortality. Nevertheless, observed variations in pregnancy–related mortality were very substantial, and highly correlated with ambulance utilisation. Since, in most of the world, women’s means of transport to health care facilities for delivery are taken for granted as an essential component of health systems, it is reasonable to suppose that the availability of transport might be just as essential in sub–Saharan Africa.The physical obstacles to reaching health facilities, and the lack of available transport options, are probably most extreme in Africa. The adjusted MMR estimate of 401 per 100 000 for Tigray in the absence of the ambulance service was consistent with international estimates of MMR for Ethiopia before ambulances were deployed , and alsIdeally this study would have been designed as an intervention with randomised allocation to ambulance transport. However, there would be very serious ethical difficulties in designing such an evaluation, given that the world in general implicitly assumes that availability of effective transportation to access obstetric services is a basic requirement, which may even be considered as a human rights issue . Given tThis operational assessment was only carried out in one Region, where survey data on maternal deaths were already available . NeverthPrevious studies of obstetric emergency transport in sub–Saharan Africa have been very limited, and none have involved non–emergency provision. A small–scale study in Burundi estimated that a large proportion of cases with complications were transferred by ambulance, but was not able to measure the effect on maternal mortality . SimilarTwo factors which might reasonably be supposed to influence pregnancy–related mortality in the absence of a free transport service were distance to District health facilities and the availability of mobile telephone networks. In Tanzania, maternal mortality and distance to hospital were strongly related . In the While extrapolating from these results might be speculative, our estimate that approximately 7000 ambulance–kilometres averted one pregnancy–related death suggests that ambulances may be a practical and cost–effective means to substantially reduce the persistently high levels of pregnancy–related mortality across sub–Saharan Africa. Assuming that a free ambulance service such as that now implemented in Ethiopia might cost around US $1 per kilometre to provide , then an annual budget of US $1 billion for free ambulance transport might avert a substantial proportion of the estimated 180 000 maternal deaths in Africa. Of course this would not simply be a matter of money–resources would have to be translated into a committed and effective transport service, which may not be easy, and local conditions and circumstances would have to be considered. But it seems clear that effective obstetric transport can make a substantial and cost–effective difference to pregnancy–related mortality–possibly achieving as much as two–thirds of the 75% reduction called for by Millennium Development Goal 5 (MDG5) .Without doubt the possibility for women to be readily transported to health facilities, both for routine deliveries and in obstetric emergencies, is a critical component of providing effective maternal care. Our results show that transport and communication innovations in Ethiopia correlated with appreciably reduced pregnancy–related mortality. This was achieved through the provision of four–wheel drive ambulances on a 24/7 basis, which could be called via the mobile telephone network. The magnitude of mortality differences correlated with ambulance utilisation amounted to a considerable proportion of the MDG5 target for maternal mortality reduction. Although this assessment only covered one Region in Ethiopia, the magnitude of the observed reduction in pregnancy–related deaths, plus the commonplace notion that women have a right to be able to physically get to maternity services, underlines the urgent necessity of considering the provision of innovative obstetric transport across Africa.


Light damages photosynthetic machinery, primarily photosystem II (PSII), and it results in photoinhibition. A new photodamage model, the two-step photodamage model, suggests that photodamage to PSII initially occurs at the oxygen evolving complex (OEC) by light energy absorbed by manganese and that the PSII reaction center is subsequently damaged by light energy absorbed by photosynthetic pigments due to the limitation of electrons to the PSII reaction center. However, it is still uncertain whether this model is applicable to photodamage to PSII under visible light as manganese absorbs visible light only weakly. In the present study, we identified the initial site of photodamage to PSII upon illumination of visible light using PSII membrane fragments isolated from spinach leaves. When PSII samples were exposed to visible light in the presence of an exogenous electron acceptor, both PSII total activity and the PSII reaction centre activity declined due to photodamage. The supplemental addition of an electron donor to the PSII reaction centre alleviated the decline of the reaction centre activity but not the PSII total activity upon the light exposure. Our results demonstrate that visible light damages OEC prior to photodamage to the PSII reaction center, consistent with two-step photodamage model. Photosynthetic organisms including plants, algae and cyanobacteria use light energy to drive the oxygenic photosynthesis but paradoxically this process is accompanied by photodamage to photosystem II (PSII). Since photosynthetic electron flow starts from the reduction of plastoquinone by electrons released by oxidation of water at PSII, the accumulation of photodamaged PSII decreases photosynthetic activity. This phenomenon is referred to as photoinhibition125in vitro using isolated PSII complexes and thylakoid membranes and in vivo using antibiotics (chloramphenicol or lincomycin) that inhibit the PSII repair though inhibiting the synthesis of the D1 protein. The rate of photodamage to PSII in the absence of PSII repair is strongly related to the intensity of incident light891012131415The process of photodamage to PSII, which can be monitored in the absence of the PSII repair process, can be studied in vivo under visible light. Recent studies also suggest that both mechanisms may occur concurrently222324There are several hypothetical mechanisms associated with photodamage to PSII518192012per se; excess energy causes photoinhibition through inhibiting the PSII repair2712131029305620The extent of photoinhibition is enhanced in conditions where the light energy absorbed by photosynthetic pigments exceeds its utilisation for photosynthesis, e.g., interruption of the Calvin-Benson cycle decreases the energy utilization capacity and accelerates photoinhibition. Therefore, it was widely assumed that excess light energy absorbed by antenna causes acceleration of photodamage to PSII through acceptor- or donor-side photoinhibitionIn the two-step photodamage model, the initial step of photodamage to PSII is not associated with the excess energy and not even light energy absorbed by photosynthetic pigments122O via functional OEC and reaction center to an artificial electron acceptor) and the PSII reaction centre activity  after PSII samples had been exposed to light in the presence of electron acceptor for PSII with or without electron donor for PSII. Our results demonstrate that an exogenous supply of electron donor for PSII alleviates the decline of PSII reaction center activity, but not PSII total activity, upon the visible light exposure. Our results show that photodamage to the PSII reaction center upon illumination with visible light is a secondary event following photodamage to the OEC. Here we propose that the two-step photodamage model is applicable to the photodamage to PSII upon illumination with visible light.In this study, we examined the initial site of photodamage to PSII under visible light using PSII membrane fragments isolated from spinach leaves. We measured the PSII total activity (electron transfer from H−2 s−1) for 30 min, the PSII activity measured by the production of oxygen declined to 10–20% of the initial activity . When PSII samples were exposed to visible light . The PSII total activity was measured by spectrophotometry. When the PSII samples were exposed to light at 1,000 μmol photons mactivity . There w−2 s−1 for 5 min in the presence of the electron acceptor PPBQ with or without electron donor diphenylcarbazide (DPC). In the absence DPC, the total PSII activity declined to 40% of initial activity after the light exposure , green  and red . PSII total activity declined to 39%, 52% and 45% of initial when PSII samples were exposed to blue, green and, red lights, respectively, in the absence of DPC  or limitation of electron donor to the excited P680 (donor-side photoinhibition) both due to excessive excitation of P680 by light absorbed by photosynthetic pigments192710In the present study, PSII samples were more sensitive to photodamage under blue and red lights than green light. This is in agreement to an earlier report of action spectrum in visible light361912In plants grown under sunlight, the photodamage to PSII happens under sunlight is largely associated with visible light272, pH 6.5), flash frozen in liquid nitrogen (LN2) and stored at −80 °C until use. Before light exposure, the sample was unfrozen and resuspended in Buffer A . In order to decrease the sucrose content, the sample was centrifuged at 24000 × g for 5 minutes and suspended in fresh Buffer A. The chlorophyll content was measured−1. The sample was kept in darkness at 4 °C at all times unless otherwise stated.PSII enriched membranes were prepared from fresh spinach leaves purchased from the market following a previous method−1) were exposed to light at 4 °C in the presence or absence of electron donor DPC (10 μM) and electron acceptor PPBQ (0.6 mM). The thickness for sample illumination was adjusted to 2.5 mm in all cases. A halogen lamp projector was used as a visible light source (for emission spectrum see PSII samples (150 μg of chlorophyll mL−1) were collected by centrifugation , resuspended in fresh Buffer A and adjusted to 7.5 μg of chlorophyll mL−1. PSII reaction center activity and PSII total activity were measured spectrophotometrically in the presence of the artificial electron acceptor 2,6-dichlorophenol indophenol (DCPIP) (1 mM) at 25 °C with or without artificial electron donor DPC (100 μM), respectively, using a UV-vis spectrophotometer . The assay was illuminated using 430 nm LED (2400 μmol of photons m−2 s−1) and the activity was monitored by absorption changes at 600 nm due to reduction of DCPIP. The detector was protected by a narrowband filter centred at 600 nm to ensure that the PMT received only the monochromatic light transmitted by the sample.After photodamage treatments, PSII samples .


Laparoscopic liver ablation therapy can be used for the treatment of primary and secondary liver malignancy. The increased incidence of cancer recurrence associated with this approach, has been attributed to the inability of monitoring the extent of ablated liver tissue.The feasibility of assessing liver ablation with probe‐based confocal laser endomicroscopy (CLE) was studied in a porcine model of laparoscopic microwave liver ablation. Following the intravenous injection of the fluorophores fluorescein and indocyanine green, CLE images were recorded at 488 nm and 660 nm wavelength and compared to liver histology. Statistical analysis was performed to assess if fluorescence intensity change can predict the presence of ablated liver tissue.CLE imaging of fluorescein at 488 nm provided good visualization of the hepatic microvasculature; whereas, CLE imaging of indocyanine green at 660 nm enabled detailed visualization of hepatic sinusoid architecture and interlobular septations. Fluorescence intensity as measured in relative fluorescence units was found to be 75–100% lower in ablated compared to healthy liver regions. General linear mixed modeling and ROC analysis found the decrease in fluorescence to be statistically significant.Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.Laparoscopic, dual wavelength CLE imaging using two different fluorophores enables clinically useful visualization of multiple liver tissue compartments, in greater detail than is possible at a single wavelength. CLE imaging may provide valuable intraoperative information on the extent of laparoscopic liver ablation. Lasers Surg. Med. 48:299–310, 2016. © 2015 The Authors.  Thermal ablation is a minimal invasive therapy, which can be used for the treatment of primary or secondary liver malignancy During thermal ablation, there is a significant change in tissue temperature which in itself can be regarded as a surrogate marker for cellular destruction in vivo histopathology. Its utilization in a variety of experimental and clinical scenarios has been increasingly reported. CLE utilizes a fiber optic element as its objective lens, allowing confocal microscopy with a micron‐scale resolution that enables visualization of subcellular details using endogenous or exogenous fluorescence. Exogenous fluorescence is provided by fluorescent dyes, which can be applied in a systemic  or topical fashion and are commonly referred to as fluorophores. They enhance CLE image contrast by absorbing and emitting light at wavelengths that are specific for each individual agent and circumstance Confocal laser endomicroscopy (CLE) is a novel optical imaging technology that enables accurate microscopic assessment of CLE during laparoscopy or NOTES  has been performed either with a rigid endomicroscope which contains the scanning optics in its tip in vivo porcine model of laparoscopic microwave liver ablation.Liver ablation has been visualized using a bench‐top confocal laser microscope in a small animal model For establishment of the model, percutaneous microwave ablation was conducted under direct laparoscopic visualization. As the CLE system used in this study has a limited imaging depth of <100 μm, only superficial ablation zones were created and assessed. Optical scattering in hepatic tissue is very high and imaging depths are likely to be no better in any other optical technique. Imaging of subsurface ablation zones was not attempted. However, imaging of deeper ablated areas is of clinical relevance, and CLE via a placed optical fiber within the periphery of an ablation volume may be a potential approach to achieving this.Previous groups evaluating laparoscopic CLE have used single fluorophores that are usually visible on CLE imaging within the blue‐light or near‐infrared spectrum Description of normal porcine liver histology on CLE examination using fluorescein at 488 nm and indocyanine green at 660 nm illumination.Evaluate the technical feasibility and limitations of CLE in an intraoperative setting.Assess the ability of CLE to predict liver tissue necrosis induced by ablation therapy.This article makes the following contributions:The experiments outlined below were performed in parallel with a different study conducted on the same animal, which has already been reported elsewhere Laparoscopic microwave ablation.Recovery period of 7–10 days.Laparoscopic CLE image acquisition (following laparoscopic liver resection in two cases).Post mortem liver biopsies.The experimental flow model is listed below, sequentially:Following approval by the local animal ethics committee, experiments were performed at Northwick Park Institute for Medical Research (London). A total of four female landrace mini‐pigs with a bodyweight ranging between 50 kg and 80 kg were studied. Each animal underwent two procedures under general anesthesia on separate days with a 7–10 day recovery period in between. On the first procedure, laparoscopic liver ablation zones were produced . During the second procedure CLE images and liver biopsies were obtained. In two animals a left laparoscopic hemihepatectomy was carried out prior to imaging, to evaluate CLE examination of the surgical resection margin.Following best practice animal husbandry, the mini‐pigs were acclimatized to their surroundings with free access to food and water at least 1 week prior to experiments. To avoid intestinal distension animals were fasted 24 hours prior to surgery but had continual access to water. Oral endotracheal intubation facilitated general anesthesia with 2% Isoflurane in oxygen in a recovery (ablation) or nonrecovery (CLE imaging) algorithm with 200 mg Pentobarbitone euthanasia at termination of surgery. Orogastric intubation decompressed the stomach during laparoscopy. All procedures were carried out with the animals in a supine position.Following establishment of the pneumoperitoneum, abdominal contents were visually inspected for potential ablation induced collateral injuries. All imaging experiments were recorded with the laparoscopic camera and stored as digital files. Position of the Cellvizio™ probe tip in relation to the ablation zones examined could be identified on video and correlated with the recorded CLE images for future analysis. Immediately before liver imaging, a standardized Cellvizio™ system calibration procedure When visualization became impeded by blood or other detritus that had attached itself to the tip of the probe, it was cleaned with normal saline. This reflects the value of an imaging scheme rather than a single fiber spectrometer. In the latter case a low signal could represent either a lack of fluorophore or a fouled fiber tip—enhancing the risk of a false reading from a given sampling site.Before experiments commenced, a training session was conducted to familiarize surgeons with CLE probe handling during laparoscopy. Particular attention was paid to probe tip pressure because too little pressure resulted in insufficient tissue contact, whereas too much pressure severely diminished fluorescence intensity. Both extremes resulted in the loss of any measurable fluorescence intensity and hence a black image which again demonstrates the value of an imaging system rather than a spectrometer fiber. The training session allowed surgeons to find a narrow range of tip pressure that enabled image acquisition at a visibly stable fluorescence level. From a surgical technique point of view, deliberate adjustment of pressure within this narrow window was very challenging, without haptic feedback so no formal quantification of probe tip pressure was undertaken. None of the image data acquired during the training session was used for analysis.Pneumoperitoneum was established using either the Veress technique or port insertion under direct vision. A periumbilical 10 mm port was used for the laparoscopic camera with additional 5 mm and 10 mm ports inserted in the right and left upper quadrants. For ablation, one additional 5 mm port was inserted in the right subcostal region. Liver resection was performed using four ports in addition to the camera port; three in the right upper and one in the left upper abdomen. Pneumoperitoneum was constantly monitored and maintained at 8–12 mm Hg. The porcine equivalent of a left hemihepatectomy was carried out without vascular inflow occlusion. A portable ultrasonic scalpel  was used for transection of the liver parenchyma. Small vessels were controlled with ultrasonic scalpel or metal clips, whereas major vessels were divided with a laparoscopic stapling device .2 fields of view.Experiments were performed sequentially in a “dual wave length approach” using both 488 nm (blue) and 660 nm (red) laser lines which were connected to separate CLE imaging systems . For CLE image acquisition 0.5–1.5 mm, flexible confocal probes  were used, all compatible with conventional endoscopic instrument channels. The CLE imaging systems control laser illumination and fiber scanning, delivering 12 frames per second which enables near real‐time image acquisition. Images at different wavelengths were recorded “in turn,” meaning without parallel coregistration of the red‐ and blue‐light laser systems. Imaging parameters were probe dependent with 1.4–3.5 μm lateral resolution, 10–30 μm axial resolution, 0–70 μm imaging depth and a 240–600 μmThe confocal probes were inserted into the peritoneal cavity through the lumen of a steerable catheter  which was itself introduced through a 5 mm laparoscopic port. The steerable catheter, designed for intravascular use, allowed subtle manipulation of the probe tip during laparoscopy Fig. a. A tech2) was too small to accurately localize with markings that would have been visible on laparoscopy.Following termination of the animals, the ablation zones and the surrounding healthy liver were resected for histological analysis. During surgery, if more than one ablation zone was examined, they were assigned numbers and their anatomical position was recorded during laparoscopy with the intention to facilitate postoperative CLE image analysis for each individual lesion. No attempt was made to perform a more detailed spatial correlation between CLE‐ and histopathological images because the CLE probe's field of view  mean fluorescence values for each image frame were recorded and exported for statistical comparison. The values were termed “relative fluorescence units” (RFU).Negative RFU values can potentially represent either image noise, or loss of autofluorescence due to prolonged illumination causing a “photo‐bleach” effect Evaluation of data distribution using the Kolmogorov–Smirnov test showed that the values in both groups were nonparametric. There was an unequal number of frame values in both groups, which would invalidate routine paired group comparison . For this reason data were analyzed employing a generalized linear mixed model (GLIMMIX) which allows for the incorporation of fixed  and random  effects. This facilitated comparison of unequally sized groups and accommodated changes in the RFU values with each new calibration of the Cellvizio™.The GLIMMIX analysis was set up to model the probability of the probe being on normal  liver parenchyma. The measured RFU value was set up as fixed effect and the animal ID  was set as a random effect in the model. To reduce data skew and kurtosis, mean values for each frame were transformed using the root‐square transformation. The GLIMMIX analysis was based on the estimation of residual marginal expansion pseudo‐likelihood. Glimmix result output is given as the change in odds ratio for an increase in RFU at a standardized value. For example, an odds ratio of 2.0 at standardized value of 100 to 101 RFU means that the odds ratio of liver parenchyma being normal increases by two if the RFU value increase by one from 100 to 101.To test the validity of the GLIMMIX model a receiver operating characteristic (ROC) analysis was performed. The covariates and intercepts provided by the GLIMMIX analysis were used to calculate the log odds, odds and finally the probability of identifying nonablated tissue. Each single probability value corresponded to a single observation (mean RFU per frame), a specific animal ID and a tissue type, which enabled us to perform a ROC analysis based on this data. Because animal ID was a random effect, the same RFU value would result in different probability values for the individual animals. This relationship explains why based on our data, no single RFU cut‐off value can be estimated as an optimal threshold to distinguish ablated from nonablated tissue.P < 0.05. Statistical analysis was performed using SPSS™ Version 21  with exception of the GLIMMIX analysis which was carried out with SAS™ Version 9.4 . Nontransformed group values are given as median and interquartile range. The workflow for the statistical analysis is shown in Figure Statistically significant differences were accepted if The histological samples obtained within 7–10 days of microwave ablation showed different degrees of cellular injury in the ablated areas. Centrally, within the ablation zone, tissue appeared macroscopically white and on histology, showed complete coagulation necrosis. Surrounding these areas was a rim of tissue that showed signs of sinusoidal obstruction, cell shrinkage and disruption of lobular architecture. On macroscopic inspection these areas were difficult to distinguish from the normal liver tissue in the periphery, validating the use of a sensitive imaging instrument for this purpose.CLE images are shown adjacent to histology sections, obtained from normal porcine liver after conclusion of the experiments. To facilitate comparison, CLE images are shown in parallel to histology slides with comparable architectural features from the same animal.When using 488 nm CLE, no auto‐fluorescence signals were recordable from the tissue before fluorescein administration. Intravenous fluorophore injection was followed by an “inflow phase” where the fluorophore is distributed and taken up by the liver parenchyma. This phase lasts for approximately 7–10 minutes after injection and is characterized by bright signals in blood vessels contrasting with dark lobule areas Fig. a and b. The inflow phase was followed by the “parenchymal phase” when the fluorescence pattern is reversed as the fluorophore has been redistributed from the vasculature into the liver parenchyma. During this phase, the vasculature is devoid of fluorescence signal and can be visualized as dark lines of increased contrast  that surrounds the liver lobules, which had by then become rich in fluorescein Fig. c and d. Similar to the fluorescein system, 660 nm CLE illumination of ICG, demonstrated no tissue auto‐fluorescence and thus no imaging of liver architecture was possible prior to fluorophore administration.In contrast to CLE of fluorescein under 488 nm excitation, it was not possible to visualize any significant fluorescence within the intravascular compartment during the “inflow phase.” The combination of ICG and 660 nm CLE, however, appeared to be better suited to imaging the sinusoid structure within liver lobules Fig. a and b. As the CLE probe has no inherent navigability, the use of an intravascular catheter  afforded free rotation, flexion, and retroflexion to 180°. Familiarity with the steering system allowed instant successful use of the CLE instrument without additional prior expertise or training.This degree of maneuverability allowed access to virtually any area on the liver surface Fig. a. The maIn ablated liver tissue, the characteristics of CLE images recorded with fluorescein and ICG were similar in all the animals studied. Whether the fluorophore was injected before or after ablation was carried out, did not affect visualization of tissue characteristics. In the majority of the ablation zone, no fluorescence was detectable which corresponded to zones of ablation induced injury on histology Fig. . When exOverall RFU values measured in nonablated versus ablated tissue decreased by 75–94% and 77–100% for CLE imaging with the fluorescein/488 nm system and the ICG/660 nm system, respectively . At this fluorophore and wavelength combination the variation introduced by imaging individual animals and recalibrating the Cellvizio™ system did not contribute to the fit of the model as a random effect with a covariance parameter of 1.66 ± 1.66 . The covariance parameter estimation indicates to what degree the random variable affects the fit of the model  of the model and its associated standard error gives a measure of repeat sampling variability. This means that the same RFU values could be applied across different animals in the study to predict the presence of healthy liver tissue. ROC analysis resulted in an area under the curve of 0.955 ± 0.003 .With CLE of fluorescein using 488 nm excitation, a standardized RFU value change from 41 to 42 (square root) increased the odds ratio of liver parenchyma being normal  by 1.16 . Variation introduced by individual animals and recalibration did significantly contribute to the fit of the model as a random effect with a covariance parameter estimation of 4.6 ± 3.8 . This means that the predictive RFU values were not interchangeable for different animals in the study as each individual's fluorophore distribution pattern within the areas of interest, were unique to that animal. ROC analysis resulted in an area under the curve of 0.999 ± 0 .A standardized RFU value change from 17 to 18 (square root) with CLE imaging of ICG at 660 nm increased the odds ratio of liver parenchyma being normal by 1.58 ; secondly, the tissue types were repeatedly measured to account for the inconsistency introduced by CLE probe movement and a small field of view; thirdly, an incongruent number of mean RFU values in each group resulted from having a different number of frames available for analysis. Lastly, the absolute values of the measured RFU were not comparable between different experimental days because each new calibration of the Cellvizio™ altered the RFU value.An analysis based on generalized linear mixed modeling has indicated that loss of fluorescence intensity can be used The general linear mixed model analysis allows for all these considerations and takes into account the correlation in measurements between different tissues in the same animal. It is also able to handle nonparametrically distributed data. A separate ROC analysis has been used to confirm the validity of the model. Before fluorescence values can be used to inform clinical decision making, however, it is imperative that standardized, absolute fluorescence values are established. If an ablation dependent variability in absolute fluorescence can be validated in more extensive datasets, it has the potential to be transformed into a computational image recognition algorithm. This algorithm could then be integrated into a clinical imaging system with the ability to monitor and map liver ablation.Each frame value was treated separately because unavoidable probe movement meant that some sections of tissue may just be represented by a single frame. Although a single image frame may not provide enough data for clinicians to evaluate tissue, it could provide sufficient data for an automatic tissue evaluation algorithm based on fluorescence intensity.A description has been provided comparing key characteristics of liver tissue architecture as imaged on fluorescein and ICG facilitated CLE examination at 488 nm and 660 nm wavelength. To account for temporal variations in imaging characteristics following fluorophore injection, a categorization into hepatic “inflow” and “parenchymal” phases has been proposed here. The inflow phase lasts approximately 7–10 minutes and takes place while the fluorophore is mainly concentrated in the hepatic vasculature. This blends over into the parenchymal phase when the bulk of fluorophore concentration has been redistributed into the intracellular compartment. Although a number of articles have evaluated CLE of the liver for different indications in animals and humans, 2 could be observed that accumulated high fluorescence intensity. Given that ICG is known to be rapidly excreted via bile In the parenchymal phase, CLE of the fluorescein/488 nm system was better suited to imaging blood vessel morphology because low contrast areas correlated better with blood vessels, whereas these areas in the ICG/660 nm system were consistent with both fibrous tissue septations and vasculature. A more homogenous and intense distribution of fluorescence throughout the liver tissue indicated that fluorescein accumulated in hepatocytes and other cells . In comparison, ICG CLE at 660 nm revealed a high affinity of ICG to hepatocytes with nuclear sparing, providing a strong contrast. Visualization of interlobular septation architecture appeared to be better suited to the 660 nm CLE system with ICG, which was able to clearly delineate liver lobules from surrounding fibrous tissue. At 30–40 minutes after ICG injection areas of 5–20 μmOverall, the detection sensitivity of ICG fluorescence intensity at 660 nm was lower when compared to fluorescein at 488 nm wavelength. This was likely due to the fact that the optical absorption maximum of ICG is 800 nm (near‐infrared spectrum) so our excitation efficiency was poorer at 660 nm, and while not optimal, was adequately within the excitation range to excite fluorescence emissions for this fluorophore. This illumination wavelength is a manufacturer preset and cannot be altered. Near‐infrared CLE imaging, however, is not available yet in a CE marked system and we felt that adequate signal was achieved in the system described, as the fluorophore has attractive properties relevant for clinical liver and bile duct imaging Although porcine models are frequently used to evaluate novel surgical technologies The relation and distribution of fluorescence characteristics in healthy and ablated liver tissue demonstrated in this article indicate that CLE may well have utility in ablation monitoring but further research is required. Before absolute RFU values can be compared between different experiments or between research groups, the Cellvizio™ platform or alternative CLE imaging systems would need to be adjusted and standardized to allow reproducible calibration of fluorescence intensity.At this stage, CLE imaging can offer micron level resolution in discriminating ablated from nonablated liver tissue and may, therefore, be of interest as a novel imaging tool in liver ablation research. If clinical translation is considered in the future, technical limitations imposed by CLE imaging depth and probe vulnerability will have to be addressed.The feasibility of utilizing needle based, diffuse optical spectroscopy to evaluate liver malignancy In the diagnosis and treatment of abdominal malignancy, there are a number of scenarios  where a needle is intentionally inserted into malignant tissue. This is feasible because the risk of needle tract seeding is either small or outweighed by the clinical benefits of the procedure Another technical issue is the CLE probe's vulnerability to high temperatures. The Cellvizio™ probes are only licensed to work within the range of normal body temperature whereas liver ablation may generate temperatures in excess of 100°C. This may prevent real time CLE monitoring within the center of an ablation zone but the temperature in the periphery of an ablated region is generally lower than in the center and rapidly deteriorates In summary, this article has assessed dual‐wavelength CLE for evaluating ablation therapy of focal liver lesions under laparoscopic guidance. Intraoperative CLE image acquisition during liver resection has been shown to be technically possible. In addition to a descriptive evaluation it was demonstrated that numerical analysis of fluorescence intensity could be employed to confirm the extent of liver ablation. Based on our findings, CLE may be of interest as a novel imaging modality in liver ablation research. Further investigation seems warranted to examine if technical limitations, that currently prevent a clinical translation of this approach, can be overcome.


SCN), where the ensemble activity of thousands of clock neurones generates and communicates circadian time cues to the rest of the brain and body. Many regions of the brain, including areas with neuroendocrine function, also contain local daily clocks that can provide feedback signals to the SCN. Although much is known about the molecular processes underpinning endogenous circadian rhythm generation in SCN neurones and, to a lesser extent, extra‐SCN cells, the electrical membrane clock that acts in partnership with the molecular clockwork to communicate circadian timing across the brain is poorly understood. The present review focuses on some circadian aspects of reproductive neuroendocrinology and processes involved in circadian rhythm communication in the SCN, aiming to identify key gaps in our knowledge of cross‐talk between our daily master clock and neuroendocrine function. The intention is to highlight our surprisingly limited understanding of their interaction in the hope that this will stimulate future work in these areas.As with many processes in nature, appropriate timing in biological systems is of paramount importance. In the neuroendocrine system, the efficacy of hormonal influence on major bodily functions, such as reproduction, metabolism and growth, relies on timely communication within and across many of the brain's homeostatic systems. The activity of these circuits is tightly orchestrated with the animal's internal physiological demands and external solar cycle by a master circadian clock. In mammals, this master clock is located in the hypothalamic suprachiasmatic nucleus ( SCN). Indeed, work spanning over 50 years is unravelling the important and intimate link between SCN activity and appropriate timing in neuroendocrine function.The developmental onset of some fundamental neuroendocrine processes of the body, such as those involved in reproductive maturity, ranges from months to years depending on the species in question, and relies on constellations of tightly regulated hormonal signalling processes within the brain and body. Once established, this timely coordinated balance in hormonal activity and reproduction is temporally regulated by our master circadian clock in the suprachiasmatic nuclei (Period1/2 (Per1/2), Cryptochrome 1/2 (Cry 1/2), Clock and Bmal1. In the past three decades, significant progress has been made to dissect and understand the intricate inner workings of this molecular timing machine that operates as a near 24‐h transcription–translation molecular feedback loop (TTFL) Circadian rhythms are daily timing in behaviour and physiology that persist (free run) in the absence of external time cues, such as light. In mammals, the master circadian clock resides in the SCN, where the activity of thousands of cell‐autonomous clock neurones is synchronised each day to output ensemble circadian time cues to the rest of the brain and body. At its most basic level, the circadian clockwork is conceptualised as an intrinsic intracellular process involving a dynamic interplay between the protein products of core clock genes such as in vivo and in vitro SCN brain slice preparations show that, at the population level, SCN neurones are significantly more active during the day (an up state: discharging APs at 4–6 Hz) than at night (a down state: generating APs at 0.1–2 Hz)  neurones of the thalamic intergeniculate leaflet, which send monosynaptic projections to the SCN via the geniculo‐hypothalamic tract Several internal physiological signals also feedback and fine tune timing precision in the SCN. Such intrinsic feedback cues  The realisation of the importance of circadian timing in hormone function started with the work of Everett and Sawyer in the 1950s. In a pioneering study, it was discovered that, in nocturnal rodents, a stimulatory drive occurring at a narrow temporal window in the pro‐oestrus afternoon is necessary for the induction of ovulation later at night et al. Indeed, when placed in constant conditions, the oestrus cycle in rodents free‐runs Perhaps the most direct evidence of an SCN‐dependent driven rhythm in hormonal secretion comes from elegant studies in female hamsters. Under normal conditions, both halves of the bilateral SCN in hamsters operate in synchrony. When these animals are housed in constant light, however, the SCN activity and behavioural rhythms ‘split’ in some hamsters, giving rise to two activity bouts in a 24‐h period Taken together, these studies demonstrate that important aspects of neuroendocrine physiology exhibit circadian variation, and that the master circadian clock is central to the timing of endocrine processes. This provides an excellent model system in which to investigate circadian and endocrine interactions.2 receptor, the preferred receptor for VIP in the SCN, is expressed throughout this nucleus Neurones of the SCN are neurochemically and functionally heterogeneous and form distinct anatomical clusters within this brain structure. Ventral SCN neurones synthesise vasoactive intestinal polypeptide (VIP), whereas the dorsal neurones contain arginine vasopressin (AVP) in vitro study has also recently shown that some intrinsic SCN clock function relies on paracrine communication amongst its neurones (mainly through diffusible neuropeptides such as VIP) in vivo studies are needed to establish the role of paracrine signalling in SCN function. When sending circadian signals to the neuroendocrine system, however, the SCN does so via direct synaptic contacts onto endocrine cells or through populations of neurones that contact endocrine cells. This suggests that, unlike daily timing in behaviour (neuromodulation) In addition to forming conventional cell‐to‐cell synaptic contacts, SCN neurones can also signal circadian timing to the body in a paracrine fashion. Indeed, the timing of behavioural rhythms in rodents, such as locomotor activity, drinking and gnawing, are under the control of the SCN paracrine tone 2 receptor is expressed by a subset of GnRH neurones in vivo and in vitro causes marked changes in GnRH, LH and gonadotrophin release 2 receptor, hypothalamic VIP expression is in phase with PRL release, and work in rodents indicates that the activity of PVN dopaminergic cells suppresses pituitary PRL secretion In the hypothalamic preoptic area (POA) of many species, VIP terminals originating from the SCN form direct contacts onto GnRH neurones, and indirectly connect to oestrodiol‐concentrating interneurones that communicate to GnRH cells V1aR) is expressed by a small population of POA GnRH neurones V1aR in the SCN is also significantly reduced There is also strong evidence in rodents suggesting that AVP signalling in the POA modulates the activity of GnRH neurones and is involved in PRL and LH secretion. AVP fibres contact GnRH neurones, and AVP secretion occurs in phase with GnRH release In the past few decades, it has also become increasingly clear that electrical and TTFL activity of SCN neurones contributes to the regulation of seasonal rhythms Taken together, these observations link the activity of the SCN, the plasticity of its neural circuits and the rhythmic release of some of its neuropeptides with the fundamental control of neurohormone secretion.The discovery and understanding of the inner workings of the TTFL come with the realisation that, although the SCN harbours the principal daily clock, many areas of the brain and body also have local circadian clockworks Per1, reveals rhythmic activity in several neuroendocrine brain regions and individual neurones, such as in dopaminergic cells that control PRL secretion Bmal1 knockout mice, the stimulating drive in GnRH release is significantly compromised Furthermore, the daily expression profile of key clock genes, such as Per1) in neuroendocrine cells remains unknown.Taken together, these observations demonstrate that the functioning of local circadian clocks in neuroendocrine tissues and at the level of the SCN is important for normal neuroendocrine function. These neuroendocrine clock circuits may not only serve as gating mechanisms that determine whether and how neuroendocrine cells respond to periodic SCN time cues, but also provide tissue‐specific local circadian time signals. This may influence output feedback signals from these tissues to the SCN Although there is limited evidence of direct neuroendocrine cell projections to the SCN Although significant recent progress in circadian neuroendocrine studies now places the activity of the newly‐identified kisspeptin and RFamide‐related peptide at the core of the communication conduits interfacing SCN signals with neuroendocrine activity Drosophila clock neurones strongly supports the concept that the electrical activity of clock neurones is integral to the functioning of the intracellular molecular clockwork in vivo chronic TTX infusion in adult animals also failed to interfere with SCN function Over the past 10 years, pioneering studies have revealed that the membrane properties, activity and excitability of SCN neurones are not only the proximal target of the TTFL, but also act to transmit information from the external world and body to the circadian molecular clockwork Figs  and 3. INevertheless, growing evidence supports the idea that information flow between the membrane and TTFL may well underlie the processes by which neurotransmitters that are released from retinal cells and the body's homeostatic systems sculpt the timing precision in the SCN. TTFL–membrane communication might also underpin how the SCN network organises excitability of its neurones to accommodate extrinsic cues to communicate circadian and seasonal signals to extra‐SCN TTFLs in the body Fig. . Our limAlthough the rate at which SCN neurones discharge APs is routinely used to report clock phase and functionality, measurement of AP firing frequency alone is not providing us with the complete picture. This is because SCN neurones also show overt day–night differences in their RMP and input resistance ] Per1‐EGFP+ve SCN neurones stop discharging APs in the middle of the day as their RMP becomes too positive (~ −30 mV) to sustain AP generation. Instead, through reducing the activity of their calcium‐activated potassium channels, these neurones enter a state of intrinsic depolarisation silencing (depolarisation blockade), producing L‐type calcium channel dependent depolarised low‐amplitude membrane oscillations (DLAMOs) instead of APs b). These modes of excitability may be important for the normal functioning of the circadian clock, and may be necessary to modulate intracellular calcium levels in SCN neurones, a key signalling molecule in SCN circadian rhythm generation processes Indeed, targeted recording of fluorescence tagged SCN neurones [from mice in which enhanced green fluorescent protein (EGFP) indicates itive ~ −0 mV to s2+]i concentration in SCN neurones. Most 2+]i levels during the day than at night. Furthermore, the concentration of [Ca2+]i reported by these studies across the day and at night is not consistent. This ranges from 191 to 440 nm during the day and from 50 to 119 nm at night. Moreover, although, under some experimental conditions, TTX abolished the day–night difference in [Ca2+]i levels 2+]i detection methods  may account for some of these discrepancies, there is no doubt that heterogeneity in SCN neurones and as yet unconfirmed excitability states of SCN cells (AP versus TTX insensitive depolarised DLAMOs) contribute significantly to these apparent inconsistencies. In support, from a single study by Hong et al. 2+]i rhythms was seen in only half of SCN neurones, leaving daily cycles of this neuronal excitability measure unaffected in others.In recent years, several studies using genetically encoded calcium sensors (e.g. GCaMP3) or synthetic fluorescent probes (e.g. Fura‐2) have documented the steady‐state intracellular calcium [Ca2+]i concentration induced by DLAMOs is yet to be described, this electrical state may be crucial for optimal daytime calcium signalling and circadian rhythm generation by clock genes 2+]i concentration during DLAMOs 2+]i concentration and calcium channel conductance values [440 nmAlthough the steady‐state [Ca2+]i levels in neuroendocrine neurones. We also have little understanding of whether and how neuroendocrine cells change their excitability to SCN and other hormonal cues over the circadian day, and whether changes in excitability of these cells can influence their genetic programmes (Fig. Taken together, these observations show that electrical signalling in the SCN is complex and that extreme depolarisation in central neurones extends beyond the borders of the SCN. This raises the possibility that these severe depolarised electrical states are more widespread than previously assumed and may be necessary for normal brain function. Studying these electrical states may reconcile some of the apparent inconsistencies seen in SCN neurophysiology and may extend our understanding of the all‐important electrical–genetic interactions in this brain structure, which may also highlight functionality in other brain circuits containing circadian clock genes. Indeed, to date, we have no records of day–night electrical excitability and [Cames Fig. .Despite our growing understanding of the cell‐autonomous processes that drive circadian rhythms in clock gene expression, our knowledge of how the molecular clockwork interacts with the membrane to drive daily changes in excitability of SCN neurones is limited. Furthermore, the nature of membrane feedback signals that convey external information to the SCN molecular clockwork is also unknown. Clock genes are found in many of the body's tissue and organ systems, and these extra‐SCN clocks are considered to be vital in providing local circadian timing in a tissue‐specific manner The observation that SCN neurones can convey circadian time cues to each other and to the body both by neural and paracrine signals demonstrates the diversity and richness of communication ‘modes’ in the SCN. Targeted recording of SCN neurones is also adding to our understanding that the electrical repertoire of SCN neurones is much broader than first appreciated. This highlights the challenges facing chronobiologists, physiologists, endocrinologists and neuroscientists in understanding circadian rhythm generation and communication in both the SCN and its targets. It is therefore prudent that, in addition to AP frequency measurements, multiple parameters of membrane excitability are assessed, preferably simultaneously, when interrogating the electrical state of SCN and extra‐SCN neurones. This will greatly increase our understanding of the processes governing cross‐talk between the master circadian clock and the neuroendocrine system, and how these signalling conduits are influenced by external cues.


The skeletal muscle system plays an important role in the independence of older adults. In this study we examine differences in the skeletal muscle transcriptome between healthy young and older subjects and (pre‐)frail older adults. Additionally, we examine the effect of resistance‐type exercise training on the muscle transcriptome in healthy older subjects and (pre‐)frail older adults.Baseline transcriptome profiles were measured in muscle biopsies collected from 53 young, 73 healthy older subjects, and 61 frail older subjects. Follow‐up samples from these frail older subjects (31 samples) and healthy older subjects (41 samples) were collected after 6 months of progressive resistance‐type exercise training. Frail older subjects trained twice per week and the healthy older subjects trained three times per week.r = −0.73).At baseline genes related to mitochondrial function and energy metabolism were differentially expressed between older and young subjects, as well as between healthy and frail older subjects. Three hundred seven genes were differentially expressed after training in both groups. Training affected expression levels of genes related to extracellular matrix, glucose metabolism ,and vascularization. Expression of genes that were modulated by exercise training was indicative of muscle strength at baseline. Genes that strongly correlated with strength belonged to the protocadherin gamma gene cluster (Our data suggest significant remaining plasticity of ageing skeletal muscle to adapt to resistance‐type exercise training. Some age‐related changes in skeletal muscle gene expression appear to be partially reversed by prolonged resistance‐type exercise training. The protocadherin gamma gene cluster may be related to muscle denervation and re‐innervation in ageing muscle. The number of people aged 65 and above has increased rapidly over the past few decades and is likely to increase progressively.Frailty and related sarcopenia are very complex, and many factors contribute to their aetiology. This includes physical inactivity, malnutrition, hormonal changes, and changes within the muscle.One of the most effective strategies to improve muscle mass and strength in adults is physical exercise.To elucidate some of these complex processes that occur in skeletal muscle during ageing, we examined the effects of prolonged resistance‐type exercise training in frail and healthy older subjects on the skeletal muscle transcriptome. By comparing genome‐level gene expression in frail and pre‐frail older subjects, healthy older subjects, and young subjects we aim to better understand the molecular causes of frailty. Secondly, we aimed to determine the effect of resistance‐type exercise training on the skeletal muscle transcriptome in both frail and healthy older people.We collected a total of 259 muscle biopsy samples from pre‐frail and frail older subjects , healthy older subjects , and young males . Some of these samples were follow‐up samples taken after 24 weeks of resistance‐type exercise training . Training for both groups was similar and consisted of progressive full‐body resistance‐type exercise training. However, the frail older group had training sessions twice per week, whereas the healthy older group trained three times per week. In addition, subjects took a protein or control drink for the duration of the study. The healthy older group received a 15 g portion of milk protein or control supplement at breakfast. The frail older group received a similar drink containing 15 g supplement drink (milk protein or control) at breakfast and lunch. More details can be found in the respective papers.et al.Biopsies from frail and pre‐frail older subjects were collected from participants of two studies performed by Tieland Musculus vastus lateralis, after local anaesthesia of the skin and fascia. Samples were freed from any visible blood and non‐muscle tissue and immediately frozen in liquid nitrogen and then stored at −80°C. All samples were obtained in the morning, in an overnight fasted state, after at least 3 days of no heavy physical activity.Muscle samples were obtained with a 5 mm Bergstrom muscle biopsy needle from the Total RNA was isolated from the skeletal muscle tissue by using Trizol reagent . Thereafter, RNA was purified using the Qiagen RNeasy Micro kit , and RNA quality was checked using an Agilent 2100 bioanalyzer . Total RNA (100 ng) was labelled using an Ambion WT expression kit  and hybridized to human whole genome Genechip Human Gene 1.1 ST arrays coding 19.732 genes, . Sample labelling, hybridization to chips, and image scanning were performed according manufacturer's instructions.P‐values were adjusted using false discovery rate.q‐value below 0.05 was considered significant. Pathway analyses were performed using Ingenuity Pathway Analysis  on the filtered dataset with the Universal exPression Codes filtering filtered genes used as the background. A sparse partial least squares (sPLS) model for leg extension 1RM  were found, with the expression of 5228 genes significantly different between young subjects and both groups of older subjects. However, fold changes of the majority of these genes were relatively subtle, which suggested small but consistent differences between these groups. Only 825 genes out of these 5228 genes showed fold changes higher than 1.2. Venn‐diagrams can be found in the Supporting Information, FigureFiguresTable FigureProlonged resistance‐type exercise training showed a significant effect on the gene expression profiles in both frail and healthy older people . Three hundred seven genes were changed in both groups after resistance‐type exercise training. Changes in expression of all these overlapping genes were in the same direction. A table with the top 20 genes changed by training is presented in Table FigureGenes related to glucose metabolism shifted away from the expression levels of the older subjects at baseline towards the levels of the younger phenotype. This trend is reflected in many other genes, where the majority of genes significantly changed by exercise training shifted towards ‘younger’ expression levels . q‐value < 0.05) we performed sPLS regression to calculate leg extension 1RM based on gene expression in the baseline samples. The aim was to evaluate whether differences in expression of the genes that were changed by training represent the overall strength of the muscle at baseline. The samples obtained after training were excluded for this analysis. A plot of the predicted leg extension 1RM strength against the measured leg extension 1RM strength is presented in FigureR2 of the model was 0.73; the mean RMSE was 17.7. The RMSE for the withheld testing set was 19.1. The top genes contributing to the model include genes from the protocadherin gamma gene cluster, CTNNBIP1, CFAP61 (C20orf26), C12orf75, and USP54. We calculated the eigengene for all protocadherin gamma genes and correlated this eigengene with leg extension 1RM. The protocadherin gamma eigengene showed a strong direct negative correlation (Pearson r = −0.73) with 1RM leg extension strength. A plot of this negative correlation is presented in the Supporting Information, FigureTo further analyse the relationship between the 307 genes that were robustly changed after training in both groups  and C12orf75. Both genes have higher expression levels in the older subjects, and training appears to attenuate expression of these genes. Not much is known about the function of CFAP61 except that it is highly expressed in skeletal muscle tissue and may be related to calcium signalling and/or energy conversion.q‐value of 0.08 after training, showing a fold change of −1.58. METTL21C encodes for a protein–lysine methyltransferase belonging to a group of proteins that are involved in methylation of chaperone proteins, where METTL21C appears to methylate HSP70 and HSP90In vitro inhibition of METTL21C expression in myoblasts showed impaired myotube differentiation and calcium signalling, suggesting that METTL21C plays an important role in the function of muscle cells and possibly also overall quality of the muscle.In our data frail subjects showed significantly higher expression levels of METTL21C compared to both the young and healthy older subjects, with a greater fold change difference between healthy and frail subjects than between the young and the older subjects Table . TraininFigureet al. state that training reverses the effect of ageing. While there is a shift towards younger expression levels, this does not necessarily mean that there is reversal of ageing. A more likely explanation is that the skeletal muscle in these older subjects have been ‘detrained’ because of more sedentary lifestyle when compared with healthy younger controls. Physical inactivity is a major contributor to age‐related muscle loss and weakness and is one of the criteria of frailty.The majority of genes that significantly changed following prolonged resistance‐type exercise training showed a shift in the expression levels towards levels observed in the younger group Figure. A previFiguresThe genes that shift towards younger expression levels include genes related to the extracellular matrix, vascularization, glucose metabolism, and muscle contraction . Both play important roles in the function of the neuromuscular junction.Our data suggest a significant remaining plasticity of ageing skeletal muscle to adapt to regular resistance‐type exercise. Many age‐related changes in skeletal muscle gene expression are partially reversed by prolonged resistance‐type exercise training. Expression of the genes robustly changed following prolonged resistance‐type exercise training in frail and healthy older subjects did not only reflect the effect of training itself but also reflected muscle strength at baseline. Expression of the protocadherin gamma gene cluster is negatively correlated with muscle strength in our data and may be related to muscle denervation and re‐innervation.We have identified a gene cluster that may be related to denervation and re‐innervation cycles in the muscle. Loss of motor neurons has been suggested to play an important role in age‐related muscle weakness and sarcopenia but is unfortunately not yet fully understood. Prolonged resistance‐type exercise training was able to modulate the expression of protocadherin gamma. Hence, studying the expression of this gene may provide novel insights on whether or not denervation and re‐innervation is modulated by interventions or lifestyle factors such as nutrition and physical activity. Furthermore, in this paper we show that steady‐state gene expression analysis provides information on the strength of the muscle itself. This suggests that it could potentially be used as a tool to provide insight into muscle strength of a subject, but theoretically also into other muscle health‐related factors.While we have a large sample size for such a study, we also have a very heterogeneous study population. The ratio of male to female among both groups of older adults is not entirely equal. We have adjusted for this in our statistical analyses where possible. Another limitation is that the muscle biopsies from the frail older adults were obtained from a study performed at Wageningen University, whereas the muscle biopsies from the healthy older adults and the young adults were obtained from studies performed at Maastricht University. Protocols for muscle biopsy collection and preparation in Wageningen are based on those from Maastricht and thus are very similar, but there may still be some bias that we cannot account for statistically. The microarray analyses were performed within the same laboratory at the same time by the same technician, which means that batch effects should be minimal. The protocols for the prolonged resistance‐type exercise training were slightly different for the frail older adults and the healthy older adults. The primary difference being that the healthy older adults trained three times per week and the frail older adults trained twice per week. As a consequence, the training stimulus for the frail older adults was somewhat lower in these individuals, and this could partially explain the decreased response among the frail older subjects. This difference in treatment also prevented us to compare the training responses in both groups directly.J.A.. Timmons is a Founding Director of XR Genomics, a Personalised Health and Fitness company. John van Duynhoven is employed by a company that manufactures and markets food products. The other authors have no competing interests to declare.Supporting info itemClick here for additional data file.Supporting info itemClick here for additional data file.Supporting info itemClick here for additional data file.Supporting info itemClick here for additional data file.Supporting info itemClick here for additional data file.Supporting info itemClick here for additional data file.Supporting info itemClick here for additional data file.Supporting info itemClick here for additional data file.Supporting info itemClick here for additional data file.Supporting info itemClick here for additional data file.


Ecology is often said to lack general theories sufficiently predictive for applications. Here, we examine the concept of a periodic table of niches and feasibility of niche classification schemes from functional trait and performance data. Niche differences and their influence on ecological patterns and processes could be revealed effectively by first performing data reduction/ordination analyses separately on matrices of trait and performance data compiled according to logical associations with five basic niche ‘dimensions’, or aspects: habitat, life history, trophic, defence and metabolic. Resultant patterns then are integrated to produce interpretable niche gradients, ordinations and classifications. Degree of scheme periodicity would depend on degrees of niche conservatism and convergence causing species clustering across multiple niche dimensions. We analysed a sample data set containing trait and performance data to contrast two approaches for producing niche schemes: species ordination within niche gradient space, and niche categorisation according to trait‐value thresholds. Creation of niche schemes useful for advancing ecological knowledge and its applications will depend on research that produces functional trait and performance datasets directly related to niche dimensions along with criteria for data standardisation and quality. As larger databases are compiled, opportunities will emerge to explore new methods for data reduction, ordination and classification. I predict there will be erected a two‐ or three‐way classification of organisms and their geometrical and temporal environments, this classification consuming most of the creative energy of ecologists. The future principles of the ecology of coexistence will then be of the form ‘for organisms of type A, in environments of structure B, such and such relations will hold.’ This is only a change in emphasis from present ecology. All successful theories, for instance in physics, have initial conditions; with different initial conditions, different things will happen. But I think initial conditions and their classification in ecology will prove to have vastly more effect on outcomes than they do in physics.Nearing the end of his life, Robert MacArthur published a book chapter in which he made some predictions about the future of ecology  of organisms (observations) among various species assemblages (populations of observations) identifies gradients in trait space and allows ordination of organisms within that space , a niche category could have phenotypic variants but still have ecological properties or functions that are essentially the same. The periodic table of elements is a concise summarisation that ignores subatomic features with little relevance for basic chemical reactivity. Ecologists deal with substantially greater variation than chemists, but could aspects be defined in a manner similar to Mendeleev's scheme? The goal of such a scheme would not be the full description of biological diversity from genes to ecosystems, but rather the organisation of species in a way that can predict ecological responses, such as invasion success or population persistence within various environmental settings, or ecological effects, such as bioturbation or nutrient cycling.et al. et al. et al. Another argument in support of periodic tables of niches is the fact that ecologists have already identified patterns of trait covariation that are strongly and consistently associated with environmental gradients , but they differ in terms of environmental gradients selecting for endpoint strategies and the suite of attributes associated with the third strategy. The C‐S‐R model distinguishes a stress‐tolerant strategy in response to stressful environmental conditions (e.g. deficit of water or nutrients) that select for a suite of functional traits described as ‘beyond K’. In contrast, the E‐P‐O model identifies a periodic endpoint strategy characterised by long lifespan, high fecundity, periodic reproduction and low investment in individual propagules favoured in habitats with large‐scale environmental variation that influences early life stage survival . Many trees, invertebrates and fishes would be classified as periodic strategists that reveal large interannual and spatial variation in recruitment.Two life history schemes that predict adaptive evolution of suites of functional traits in response to selection imposed by abiotic and biotic environmental factors are the ts Grime , and EquC‐S‐R model has proven useful for interpreting local species assemblage patterns in plants and other groups, including soil invertebrates and corals . Simple examples for adaptation to habitat structure are animals that require certain kinds of substrates for probing and extracting invertebrate prey: fishes , birds  and mammals . For most kinds of organisms, we already have identified numerous functional traits and performance measures that directly influence fitness along habitat gradients defined according to abiotic and structural environmental features.For any organism to survive, it must occupy a suitable abiotic environment with conditions within its tolerance limits. More often than not, the organism inhabits an environment that maximises fitness or surplus energy relative to metabolic demands . Functional constraints define these solutions, and extensive convergent evolution is therefore anticipated, and indeed is observed. Full development of a trophic dimension also would be fundamental, because all organisms must acquire and assimilate resources for maintenance, growth and reproduction. Trophic guilds are one of the most intuitive concepts for grouping animals according to functional similarity. Trade‐offs involved in feeding mechanics  and foraging strategies (e.g. sit‐and‐wait ambushers vs. active wide‐ranging searchers) has produced extensive convergent evolution among trophic niches in most major animal groups, and stoichiometric gradients have been used to arrange plants, microbes and herbivorous insects within nutritional niche space . Morphology sometimes fails to match predictions about performance due to behavioural plasticity or many‐to‐one mapping whereby different morphologies are functionally similar. In addition, behavioural and biochemical traits influencing and responding to indirect species interactions, such as facilitation, could be difficult to incorporate into a multidimensional niche scheme.Obviously, the simplest form would be a two‐dimensional matrix similar to the periodic table of elements, with columns being trophic niches and rows representing life history strategies Pianka  or columet al. et al. et al. Any niche scheme assumes that certain species cluster around adaptive peaks defined by sets of trait combinations associated with a given set of environmental attributes within a given type of habitat and biome. Using multivariate methods of dimension‐reduction, the universe of possible trait combinations can be reduced to reveal sets of realised combinations corresponding to adaptive peaks in trait space  – not a posteriori .Analyses aimed at producing a niche scheme should first divide the database of functional traits and performance measures into subsets based on their logical associations with each of five fundamental niche dimensions. Analyses then would be performed for each niche dimension separately, after which separate dimensional ordinations are integrated to produce interpretable niche schemes  studied extensively by KOW. Data were obtained for 56 common fish species; an additional 33 species were collected in numbers insufficient to yield reliable data, and were excluded. Data were obtained for variables associated with five fundamental niche dimensions: habitat use, life history strategy, trophic ecology, defence, and metabolism/physiology. The five data sets (Tables S2‐7) collectively contained 38 variables pertaining to functional traits or ecological performance.et al. PCA was performed on each dataset based on eigenanalysis of the correlation matrix using the VEGAN package in R 3.1.0 . Ordination of species scores for this ‘PCA of PCAs’ (Table S13) represents a two‐dimensional continuum integrating patterns (strategies) within each of the five niche dimensions. Consequently, the five niche dimensions estimated by the five data sets have an equal chance to influence the overall niche gradients and species ordinations. Interpretation of these gradients is necessarily dependent upon interpretations of gradients obtained previously from PCAs of the five original data sets. The PCA of PCAs produced a dominant gradient that contrasted inactive, armoured, benthic fishes with surface‐oriented fishes that were active swimmers feeding on invertebrates, and a secondary gradient contrasting diverse microphagous species with opportunistic life history strategies and limited capacity for energy storage vs. large predators with equilibrium life history strategies.Hoplias malabaricus (Bloch) – 2,4,2,1,3). The full dendrogram generated from this assemblage of 56 fishes has 1440 potential terminal nodes (the product of the number of groupings for each dimension). Only 50 of these potential nodes were occupied, with most potential trait combinations either non‐viable or vacant, and certain niches occupied by multiple species. For example, Ctenobrycon spilurus  and Pyrrhulina lugubris Eigenmann occupied a common niche associated with omnivory, rapid and sustained movement during foraging and predator escape, high energy expenditure, limited fat storage and an opportunistic life history strategy. The number of terminal nodes depends on criteria chosen for clustering; nonetheless, this approach is useful for detection of vacant niches. Some trait combinations are unobserved because they are non‐viable , whereas others may be viable but not present with a given species assemblage or evolutionary lineage. The latter could occur for a host of reasons, including evolutionary or biogeographic contingencies  or ecological factors .For a niche classification scheme, dendrograms from CART analysis of the five data sets  were combined in a hierarchy to construct a composite tree  analysis), each of which will produce slightly different results. Criteria for grouping species affect the scheme's resolution; therefore, this approach must be guided by the intended applications of the classification and grounded in sound knowledge of the taxa. Quality assurance measures are required to ensure data are reliable with consistent scale and resolution to enable reasonable interspecific comparisons. Criteria for pruning regression trees and other methods are available to reduce subjectivity associated with clustering algorithms. As an example, we performed CART analysis using as response variables the species scores on the first two principal components derived from PCA of data sets compiled for each of five niche dimensions, using original trait and performance values as explanatory variables to create dendrograms Fig. . GroupinContinuous niche schemes Fig.  facilitaa priori if any single trait or dimension had a disproportionate influence. Highest correlation was between the coefficient of variation in abundance and PC1 from the PCA of PCAs (0.28), and the next highest correlation was between abundance and PC2 from the PCA of PCAs (0.18). By comparison, the same analysis performed using PC scores from the PCA performed using raw data for all 38 trait variables (Table S23) yielded lower correlations with the coefficient of variation in abundance (0.21 with PC2) and abundance (0.10 with PC1). Although much residual variation remained in both cases, the PCA of PCAs appears to describe relationships between niches and ecological responses more effectively.To explore the potential usefulness of our proposed approach, we compiled a data set containing mean abundance of each fish species at Caño Maraca based on 12 consecutive monthly surveys (Table S7) for comparison with species scores on the first two PC axes of each of five niche dimensions, and with species scores on first two axes from the PCA of the PCAs as described in Box Gradients derived from the PCA of PCAs seem to have more robust interpretations compared to those derived from the PCA performed using the data set of raw values for 38 traits  within a fundamental niche dimension could be obscured by inclusion of large numbers of ecologically relevant, correlated, yet functionally unrelated traits. For example, it is unlikely that life history strategies and their influence on population dynamics would be perceived in results from analysis of a large data set that combines just a few life history traits  with a much larger collection of traits associated with other niche dimensions.A major goal in invasion biology is to identify sets of traits that may be used to forecast which species have greatest potential to be invasive (van Kleunen Biological assessment frequently evaluates how the structure of impacted communities deviates from that of the pre‐impact natural community. Patrick  pioneereet al. et al. et al. et al. et al. n‐dimensional niche. A functionally equivalent seed disperser may or may not be equally capable of defence or demographic resilience to local patterns of environmental disturbances. A general niche scheme could enhance assessment of ecological restoration proposals.Some contend that restoration is a proving ground for ecological understanding (Young Some might argue that ecology has no analogy with the periodic table of elements and that any attempt to create a general niche scheme is bound to fail Steffen . We disaet al. et al. Perhaps in the future, ecology will develop sufficient understanding of species niches and community structure to predict responses to environmental impacts and restoration efforts. The feasibility of niche schemes ultimately will depend on how we address some basic challenges. We need more rigorous criteria for identifying traits directly related to different niche dimensions (Blondel Our goal here was to demonstrate the potential feasibility of a general niche scheme while exploring some conceptual and methodological issues. The ultimate test of such schemes will be their predictive capabilities and degree of utility in ecological applications. A universal periodic table of niches is unlikely, but instead alternative niche schemes could be developed for making predictions for different groups of organisms in different regions, or for addressing different kinds of problems.But the science of ecology finally has some structure, even if not a very orderly structure as yet, and it is from the shortcomings of its present structure that we can make the safest predictions of the future.’ …. ‘Perhaps niche will turn out to be a concept that requires some subdivision into several precise definitions.In his 1972 chapter, Robert MacArthur offered the following insights:et al. MacArthur's niche classification could be considered a metaphor for the direction ecology has taken over recent decades, and it might one day be possible to organize species according to an orderly structure. Actually, this is already happening – ecology is now strongly focused on functional traits, their patterns of constraint, and their relationship with environmental variables and community structure (Cadotte KOW and ERP developed initial concepts; all authors reviewed the literature; and KOW, DBF and LMB compiled data, performed analyses and prepared figures, tables and appendices. KOW wrote the first draft of the manuscript, and all authors contributed substantially to revisions. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.