A minimum of two distinct heritable factors are required to explain correlation structures in proliferating lymphocytes

During the adaptive immune response, lymphocyte populations undergo a characteristic three-phase process: expansion through a series of cell divisions; cessation of expansion; and, finally, most of the accumulated lymphocytes die by apoptosis. The data used, thus far, to inform understanding of these processes, both in vitro and in vivo, are taken from flow cytometry experiments. One significant drawback of flow cytometry is that individual cells cannot be tracked, so that it is not possible to investigate interdependencies in the fate of cells within a family tree. This deficit in experimental information has recently been overcome by Hawkins et al. (Hawkins et al. 2009 Proc. Natl Acad. Sci. USA 106, 13 457–13 462 (doi:10.1073/pnas.0905629106)), who reported on time-lapse microscopy experiments in which B-cells were stimulated through the TLR-9 receptor. Cells stimulated in this way do not aggregate, so that data regarding family trees can be recorded. In this article, we further investigate the Hawkins et al. data. Our conclusions are striking: in order to explain the familial correlation structure in division times, death times and propensity to divide, a minimum of two distinct heritable factors are necessary. As the data show that two distinct factors are necessary, we develop a stochastic model that has two heritable factors and demonstrate that it can reproduce the key features of the data. This model shows that two heritable factors are sufficient. These deductions have a clear impact upon biological understanding of the adaptive immune response. They also necessitate changes to the fundamental premises behind the tools developed by statisticians to draw deductions from flow cytometry data. Finally, they affect the mathematical modelling paradigms that are used to study these systems, as these are widely developed based on assumptions of cellular independence that are not accurate.     

Homological scaffolds of brain functional networks

Networks, as efficient representations of complex systems, have appealed to scientists for a long time and now permeate many areas of science, including neuroimaging (Bullmore and Sporns 2009 Nat. Rev. Neurosci. 10, 186–198. (doi:10.1038/nrn2618)). Traditionally, the structure of complex networks has been studied through their statistical properties and metrics concerned with node and link properties, e.g. degree-distribution, node centrality and modularity. Here, we study the characteristics of functional brain networks at the mesoscopic level from a novel perspective that highlights the role of inhomogeneities in the fabric of functional connections. This can be done by focusing on the features of a set of topological objects—homological cycles—associated with the weighted functional network. We leverage the detected topological information to define the homological scaffolds, a new set of objects designed to represent compactly the homological features of the correlation network and simultaneously make their homological properties amenable to networks theoretical methods. As a proof of principle, we apply these tools to compare resting-state functional brain activity in 15 healthy volunteers after intravenous infusion of placebo and psilocybin—the main psychoactive component of magic mushrooms. The results show that the homological structure of the brain''s functional patterns undergoes a dramatic change post-psilocybin, characterized by the appearance of many transient structures of low stability and of a small number of persistent ones that are not observed in the case of placebo.     

The furrows of Rhinolophidae revisited

Rhinolophidae, a family of echolocating bats, feature very baroque noseleaves that are assumed to shape their emission beam. Zhuang & Muller (Zhuang & Muller 2006 Phys. Rev. Lett. 97, 218701 (doi:10.1103/PhysRevLett.97.218701); Zhuang & Muller 2007 Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 76(Pt. 1), 051902 (doi:10.1103/PhysRevE.76.051902)) have proposed, based on finite element simulations, that the furrows present in the noseleaves of these bats act as resonance cavities. Using Rhinolophus rouxi as a model species, they reported that a resonance phenomenon causes the main beam to be elongated at a particular narrow frequency range. Virtually filling the furrows reduced the extent of the main lobe. However, the results of Zhuang & Muller are difficult to reconcile with the ecological background of R. rouxi. In this report, we replicate the study of Zhuang & Muller, and extend it in important ways: (i) we take the filtering of the moving pinnae into account, (ii) we use a model of the echolocation task faced by Rhinolophidae to estimate the effect of any alterations to the emission beam on the echolocation performance of the bat, and (iii) we validate our simulations using a physical mock-up of the morphology of R. rouxi. In contrast to Zhuang & Muller, we find the furrows to focus the emitted energy across the whole range of frequencies contained in the calls of R. rouxi (both in simulations and in measurements). Depending on the frequency, the focusing effect of the furrows has different consequences for the estimated echolocation performance. We argue that the furrows act to focus the beam in order to reduce the influence of clutter echoes.     

Air motion sensing hairs of arthropods detect high frequencies at near-maximal mechanical efficiency

Using measurements based on particle image velocimetry in combination with a novel compact theoretical framework to describe hair mechanics, we found that spider and cricket air motion sensing hairs work close to the physical limit of sensitivity and energy transmission in a broad range of relatively high frequencies. In this range, the hairs closely follow the motion of the incoming flow because a minimum of energy is dissipated by forces acting in their basal articulation. This frequency band is located beyond the frequency at which the angular displacement of the hair is maximum which is between about 40 and 600 Hz, depending on hair length (Barth et al. [1] Phil. Trans. R. Soc. Lond. B 340, 445–461 (doi:10.1098/rstb.1993.0084)). Given that the magnitude of natural airborne signals is known to decrease with frequency, our results point towards the possible existence of spectral signatures in the higher frequency range that may be weak but of biological significance.     

Transmission models indicate Ebola virus persistence in non-human primate populations is unlikely

Infectious diseases that kill their hosts may persist locally only if transmission is appropriately balanced by susceptible recruitment. Great apes die of Ebola virus disease (EVD) and have transmitted ebolaviruses to people. However, understanding the role that apes and other non-human primates play in maintaining ebolaviruses in Nature is hampered by a lack of data. Recent serological findings suggest that few non-human primates have antibodies to EVD-causing viruses throughout tropical Africa, suggesting low transmission rates and/or high EVD mortality (Ayouba A et al. 2019 J. Infect. Dis. 220, 1599–1608 (doi:10.1093/infdis/jiz006); Mombo IM et al. 2020 Viruses 12, 1347 (doi:10.3390/v12121347)). Here, stochastic transmission models of EVD in non-human primates assuming high case-fatality probabilities and experimentally observed or field-observed parameters did not allow viral persistence, suggesting that non-human primate populations are highly unlikely to sustain EVD-causing infection for prolonged periods. Repeated introductions led to declining population sizes, similar to field observations of apes, but not viral persistence.     

Unified model of short- and long-term HIV viral rebound for clinical trial planning

Antiretroviral therapy (ART) effectively controls HIV infection, suppressing HIV viral loads. Typically suspension of therapy is rapidly followed by rebound of viral loads to high, pre-therapy levels. Indeed, a recent study showed that approximately 90% of treatment interruption study participants show viral rebound within at most a few months of therapy suspension, but the remaining 10%, showed viral rebound some months, or years, after ART suspension. Some may even never rebound. We investigate and compare branching process models aimed at gaining insight into these viral dynamics. Specifically, we provide a theory that explains both short- and long-term viral rebounds, and post-treatment control, via a multitype branching process with time-inhomogeneous rates, validated with data from Li et al. (Li et al. 2016 AIDS 30, 343–353. (doi:10.1097/QAD.0000000000000953)). We discuss the associated biological interpretation and implications of our best-fit model. To test the effectiveness of an experimental intervention in delaying or preventing rebound, the standard practice is to suspend therapy and monitor the study participants for rebound. We close with a discussion of an important application of our modelling in the design of such clinical trials.     

Designing an optimal water quality monitoring network for river systems using constrained discrete optimization via simulation

The problem of designing a water quality monitoring network for river systems is to find the optimal location of a finite number of monitoring devices that minimizes the expected detection time of a contaminant spill event while guaranteeing good detection reliability. When uncertainties in spill and rain events are considered, both the expected detection time and detection reliability need to be estimated by stochastic simulation. This problem is formulated as a stochastic discrete optimization via simulation (OvS) problem on the expected detection time with a stochastic constraint on detection reliability; and it is solved with an OvS algorithm combined with a recently proposed method called penalty function with memory (PFM). The performance of the algorithm is tested on the Altamaha River and compared with that of a genetic algorithm due to Telci, Nam, Guan and Aral (2009) Telci, I. T., K. Nam, J. Guan, and M.M. Aral, 2009. “Optimal Water Quality Monitoring Network Design for River Systems.” Journal of Environmental Management, 90 (3–4): 2987–2998. doi: 10.1016/j.jenvman.2009.04.011[Crossref], [PubMed], [Web of Science ®] , [Google Scholar].     

Supercritical Antisolvent Process Applied to the Pharmaceutical Industry

Pharmaceutical preparations are the final product of a technological process that gives the drugs the characteristics appropriate for easy administration, proper dosage, and enhancement of the therapeutic efficacy. The design of pharmaceutical preparations in nanoparticulate form has emerged as a new strategy for drug delivery (Pasquali, Bettini, and Giordano, 2006 Pasquali , I. , R. Bettini , and F. Giordano . 2006 . Solid-state chemistry and particle engineering with supercritical fluids in pharmaceutics . Eur. J. Pharm. Sci. 27 : 299 – 310 .[Crossref], [PubMed], [Web of Science ®] , [Google Scholar]). Particle size (PS) and particle size distribution (PSD) are critical parameters that determine the rate of dissolution of the drug in the biological fluids and, hence, have a significant effect on the bioavailability of those drugs that have poor solubility in water, for which the dissolution is the rate-limiting step in the absorption process (Perrut, Jung, and Leboeuf, 2005 Perrut , M. , J. Jung , and F. Leboeuf . 2005 . Enhancement of dissolution rate of poorly-soluble active ingredients by supercritical fluid processes: Part I: Micronization of neat particles . Int. J. Pharm. 288 : 3 – 10 .[Crossref], [PubMed], [Web of Science ®] , [Google Scholar]; Van Nijlen et al., 2003 Van Nijlen , T., G. Van Den Mooter , R. Kinget , P. Augustijns , N. Blaton , and K. Brennan . 2003 . Improvement of the dissolution rate of artemisinin by means of supercritical fluid technology and solid dispersions . Int. J. Pharm. 254 : 173 – 181 .[Crossref], [PubMed], [Web of Science ®] , [Google Scholar]). Supercritical antisolvent (SAS) processes have been widely used to precipitate active pharmaceutical ingredients (APIs) (Chattopadhyay and Gupta, 2001 Chattopadhyay , P. , and R. B. Gupta . 2001 . Production of antibiotic nanoparticles using supercritical CO₂ as antisolvent with enhanced mass transfer . Ind. Eng. Chem. Res. 40 : 3530 – 3539 .[Crossref], [Web of Science ®] , [Google Scholar]; Rehman et al., 2001 Rehman , M. , B. Y. Shekunov , P. York , and P. Colthorpe . 2001 . Solubility and precipitation of nicotinic acid in supercritical carbon dioxide . J. Pharm. Sci. 90 : 1570 – 1582 .[Crossref], [PubMed], [Web of Science ®] , [Google Scholar]) with a high level of purity, suitable dimensional characteristics, narrow PSD, and spherical morphologies. The SAS process is based on the particular properties of the supercritical fluids (SCFs). These fluids have diffusivities two orders of magnitude larger than those of liquids, resulting in a faster mass transfer rate SCF properties (solvent power and selectivity) can be also adjusted continuously by altering the experimental conditions (temperature and pressure). As a consequence, SCFs can be removed from the process by a simple change from the supercritical to room conditions, which avoids difficult post-treatments of waste liquid streams. Carbon dioxide (CO₂) at supercritical conditions, among all possible SCFs, is largely used because of its relatively low critical temperature (31.1°C) and pressure (73.8 bar), low toxicity, and low cost. In this article, we show some results about processed antibiotics (ampicillin and amoxicillin), two of the world's most widely prescribed antibiotics, when they are dissolved in 1-methyl-2-pyrrolidone (NMP) and carbon dioxide is used as antisolvent.     

Experimental evaluation of the viscoelasticity of porcine vitreous

This study aims to estimate the material properties of the porcine vitreous while testing it in close to its natural physiological conditions. Eighteen porcine eyes were tested within 48 h post-mortem. A custom-built computer-controlled test rig was designed to support, load and monitor the behaviour of eye globes while being subjected to dynamic rotation cycles mimicking saccade eye movement. Specimens were glued to the base of a container, surrounded by gelatin, frozen and cut in half to expose the vitreous. After thawing, the container was subjected to concentric dynamic rotations of up to 5°, 10° or 15°, while taking 50 MP photos of the specimen every 2 ms. The images were analysed by a digital image correlation algorithm to trace the movement of marked points on the vitreous surface with different radii from the centre of the posterior chamber. The initial camera image was used in building a finite-element model of the test set-up, which was used in an inverse analysis exercise to estimate the material properties of the vitreous. Angular displacements of the monitored points were up to 3.3°, 4.1° and 3.9° in response to eye rotations of 5°, 10° and 15°, respectively. With the experimental relationships between eye rotation and angular displacements used as target behaviour, the inverse analysis exercise estimated the initial shear modulus, the long-term shear modulus and the viscoelastic decay constant of the porcine vitreous as 2.10 ± 0.15 Pa, 0.50 ± 0.04 Pa and 1.20 ± 0.09 s⁻¹, respectively. Consideration of the viscoelasticity of the vitreous was essential to represent its experimental behaviour. Testing the vitreous in close to its normal physiological conditions produced estimations of the initial shear modulus and long-term shear modulus that were, respectively, smaller and larger than reported values (Zimberlin et al. 2010 Soft Matter 6, 3632–3635. (doi:10.1039/b925407b), Liu et al. 2013 J. Biomech. 46, 1321–7. (doi:10.1016/j.jbiomech.2013.02.006), Rossi et al. 2011 Invest. Ophthalmol. Vis. Sci. 52, 3994–4002. (doi:10.1167/iovs.10-6477)).     

Modelling the layer-specific three-dimensional residual stresses in arteries,with an application to the human aorta

This paper provides the first analysis of the three-dimensional state of residual stress and stretch in an artery wall consisting of three layers (intima, media and adventitia), modelled as a circular cylindrical tube. The analysis is based on experimental results on human aortas with non-atherosclerotic intimal thickening documented in a recent paper by Holzapfel et al. ( Holzapfel et al. 2007 Ann. Biomed. Eng. 35, 530–545 (doi:10.1007/s10439-006-9252-z)). The intima is included in the analysis because it has significant thickness and load-bearing capacity, unlike in a young, healthy human aorta. The mathematical model takes account of bending and stretching in both the circumferential and axial directions in each layer of the wall. Previous analysis of residual stress was essentially based on a simple application of the opening-angle method, which cannot accommodate the three-dimensional residual stretch and stress states observed in experiments. The geometry and nonlinear kinematics of the intima, media and adventitia are derived and the associated stress components determined explicitly using the nonlinear theory of elasticity. The theoretical results are then combined with the mean numerical values of the geometrical parameters and material constants from the experiments to illustrate the three-dimensional distributions of the stretches and stresses throughout the wall. The results highlight the compressive nature of the circumferential stress in the intima, which may be associated with buckling of the intima and its delamination from the media, and show that the qualitative features of the stretch and stress distributions in the media and adventitia are unaffected by the presence or absence of the intima. The circumferential residual stress in the intima increases significantly as the associated residual deformation in the intima increases while the corresponding stress in the media (which is compressive at its inner boundary and tensile at its outer boundary) is only slightly affected. The theoretical framework developed herein enables the state of residual stress to be calculated directly, serves to improve insight into the mechanical response of an unloaded artery wall and can be extended to accommodate more general geometries, kinematics and states of residual stress as well as more general constitutive models.     

Quantification of the passive and active biaxial mechanical behaviour and microstructural organization of rat thoracic ducts

Mechanical loading conditions are likely to play a key role in passive and active (contractile) behaviour of lymphatic vessels. The development of a microstructurally motivated model of lymphatic tissue is necessary for quantification of mechanically mediated maladaptive remodelling in the lymphatic vasculature. Towards this end, we performed cylindrical biaxial testing of Sprague–Dawley rat thoracic ducts (n = 6) and constitutive modelling to characterize their mechanical behaviour. Spontaneous contraction was quantified at transmural pressures of 3, 6 and 9 cmH₂O. Cyclic inflation in calcium-free saline was performed at fixed axial stretches between 1.30 and 1.60, while recording pressure, outer diameter and axial force. A microstructurally motivated four-fibre family constitutive model originally proposed by Holzapfel et al. (Holzapfel et al. 2000 J. Elast. 61, 1–48. (doi:10.1023/A:1010835316564)) was used to quantify the passive mechanical response, and the model of Rachev and Hayashi was used to quantify the active (contractile) mechanical response. The average error between data and theory was 8.9 ± 0.8% for passive data and 6.6 ± 2.6% and 6.8 ± 3.4% for the systolic and basal conditions, respectively, for active data. Multi-photon microscopy was performed to quantify vessel wall thickness (32.2 ± 1.60 µm) and elastin and collagen organization for three loading conditions. Elastin exhibited structural ‘fibre families’ oriented nearly circumferentially and axially. Sample-to-sample variation was observed in collagen fibre distributions, which were often non-axisymmetric, suggesting material asymmetry. In closure, this paper presents a microstructurally motivated model that accurately captures the biaxial active and passive mechanical behaviour in lymphatics and offers potential for future research to identify parameters contributing to mechanically mediated disease development.     

Prediction of Concentration Distribution in Pipeline Flow of Highly Concentrated Slurry

The Kaushal and Tomita's (2002a Kaushal , D. R. , and Y. Tomita . 2002a . An improved method for predicting pressure drop along slurry pipeline . Particulate Science and Technology 20 : 1 – 20 .[Taylor & Francis Online] , [Google Scholar]) model, which has already been found satisfactory for broadly graded multisized particulate zinc tailings slurry with moderate concentration up to 26%, flow velocity up to 3.5 m/s in 105 mm diameter pipe, mean diameter 140 μm, and geometric standard deviation of particles of 4.0, is tested for concentration distribution data collected by Kaushal et al. (2005 Kaushal , D. R. , K. Sato , T. Toyota , K. Funatsu , and Y. Tomita . 2005 . Effect of particle size distribution on pressure drop and concentration profile in pipeline flow of highly concentrated slurry . International Journal of Multiphase Flow. 31 : 809 – 823 .[Crossref], [Web of Science ®] , [Google Scholar]) on two sizes of glass beads, of which mean diameter and geometric standard deviation are 440 μm and 1.2, and 125 μm and 1.15, respectively, with concentration up to 50% and flow velocity up to 5 m/s in 54.9 mm diameter pipe. Kaushal and Tomita's (2002a) model gives more asymmetric concentration distributions. A modified model is proposed by alleviating some of the restrictive assumptions used in the existing model. Comparison of experimental data by Kaushal et al. (2005 Kaushal , D. R. , K. Sato , T. Toyota , K. Funatsu , and Y. Tomita . 2005 . Effect of particle size distribution on pressure drop and concentration profile in pipeline flow of highly concentrated slurry . International Journal of Multiphase Flow. 31 : 809 – 823 .[Crossref], [Web of Science ®] , [Google Scholar]), Gillies and Shook (1994 Gillies , R. G. , and C. A. Shook . 1994 . Concentration distributions of sand slurries in horizontal pipe flow . Particulate Science Technology 12 : 45 – 69 .[Taylor & Francis Online], [Web of Science ®] , [Google Scholar]), and Matousek (2009 Matousek , V. 2009 . Concentration profiles and solids transport above stationary deposit in enclosed conduit . Journal of Hydraulic Engineering 135 : 1101 – 1106 .[Crossref], [Web of Science ®] , [Google Scholar]) with the proposed model is satisfactory.     

Algorithm to identify the optimal perturbation based on the net basin‐of‐state of perturbed states in Boolean network

Boolean networks are widely used to model gene regulatory networks and to design therapeutic intervention strategies to affect the long‐term behavior of systems. Here, the authors investigate the 1 bit perturbation, which falls under the category of structural intervention. The authors’ idea is that, if and only if a perturbed state evolves from a desirable attractor to an undesirable attractor or from an undesirable attractor to a desirable attractor, then the size of basin of attractor of a desirable attractor may decrease or increase. In this case, if the authors obtain the net BOS of the perturbed states, they can quickly obtain the optimal 1 bit perturbation by finding the maximum value of perturbation gain. Results from both synthetic and real biological networks show that the proposed algorithm is not only simpler and but also performs better than the previous basin‐of‐states (BOS)‐based algorithm by Hu et al..Inspec keywords: perturbation theory, genetics, Boolean functionsOther keywords: optimal perturbation, perturbed states, Boolean network, gene regulatory networks, basin‐of‐states‐based algorithm, state‐transition diagram, structural intervention, perturbation gain, synthetic biological networks, real biological networks, 1 bit perturbation     

Lagrangian relaxation based heuristic for an integrated production and maintenance planning problem

In this paper, an approach is developed to solve the joint production planning and maintenance problem. Moreover, some propositions and mathematical properties were suggested and applied in the proposed heuristic to solve this integrated problem. It is based on Lagrangian relaxation (Fisher, M.L., 1981 Fisher, M.L. 1981. Lagrangian relaxation method for solving integer programming problem. Management Science, 27(1): 1–18. [Crossref], [Web of Science ®] , [Google Scholar]. Lagrangian relaxation method for solving integer programming problem. Management Science, 27 (1), 1–18) of the capacity constraints and sub-gradient optimisation. At every step of the sub-gradient method, a smoothing procedure is applied to the solution of the Lagrangian problem to ensure the feasibility of solution and to improve it. Computational experiments are carried out to show the results obtained by our approaches and are compared to those of a commercial solver.     

Base Pressure Measurements under a Scale Model Stockpile: Effects of Offset Reclaim Channels and Predictive Model Developments

It has been shown (McBride, 2006 McBride , W. 2006 . Base pressure measurements under a scale model stockpile . Part. Sci. Technol. 24 : 59 – 71 .[Taylor & Francis Online], [Web of Science ®] , [Google Scholar]) that under a scale model stockpile a central pressure dip, or ‘M’ pressure, is formed during both the filling and refilling processes of a stockpile containing a central reclaim channel. This article presents the results from further experiments with a noncentral reclaim channel and clearly illustrates that a central pressure dip exists under a conical stockpile with an offset reclaim channel. Additional data were recorded regarding lateral load transfer within the stockpile on initiation of flow, although more experimental work is required to fully understand this aspect. A method for predicting conical stockpile base pressures is presented along with an overpressure theory to account for the vertical load instability induced by the onset of material discharge.     

Protein abundance may regulate sensitivity to external cues in polarized cells

Cell polarization is a ubiquitous process which results in cellular constituents being organized into discrete intracellular spatial domains. It occurs in a variety of cell types, including epithelial cells, immune system cells and neurons. A key player in this process is the Par protein family whose asymmetric localization to anterior and posterior parts of the cell is crucial for proper division and cell fate specification. In this paper, we explore a stochastic analogue of the temporal model of Par protein interactions first developed in Dawes & Munro (Dawes and Munro 2011 Biophys. J. 101, 1412–1422. (doi:10.1016/j.bpj.2011.07.030)). We focus on how protein abundance influences the behaviour of both the deterministic and stochastic versions of the model. In Dawes & Munro (2011), it was found that bistable behaviour in the temporal model of Par protein led to the existence of complementary domains in the corresponding spatio-temporal model. Here, we find that the corresponding temporal stochastic model permits switching behaviour (the model solution ‘jumps’ between steady states) for lower protein abundances, whereas for higher protein abundances the stochastic and deterministic models are in good agreement (the model solution evolves to one of two steady states). This led us to the testable hypothesis that cells with lower abundances of Par protein may be more sensitive to external cues, whereas cells with higher abundances of Par protein may be less sensitive to external cues. In order to gain more control over the precise abundance of Par protein, we proposed and explored a second model (again, examining both deterministic and stochastic versions) in which the total number of Par molecules is conserved. We found that this model required an additional dimerization reaction in the cytoplasm in order for bistable and switching behaviour to be found. Once this additional reaction was included, we found that both the first and second models gave qualitatively similar results but in different regions of the parameter space, suggesting a further regulatory mechanism that cells could potentially use to modulate their response to external signals.     

Superstatistical modelling of protein diffusion dynamics in bacteria

A recent experiment (Sadoon AA, Wang Y. 2018 Phys. Rev. E 98, 042411. (doi:10.1103/PhysRevE.98.042411)) has revealed that nucleoid-associated proteins (i.e. DNA-binding proteins) exhibit highly heterogeneous diffusion processes in bacteria where not only the diffusion constant but also the anomalous diffusion exponent fluctuates for the various proteins. The distribution of displacements of such proteins is observed to take a q-Gaussian form, which decays as a power law. Here, a statistical model is developed for the diffusive motion of the proteins within the bacterium, based on a superstatistics with two variables. This model hierarchically takes into account the joint fluctuations of both the anomalous diffusion exponents and the diffusion constants. A fractional Brownian motion is discussed as a possible local model. Good agreement with the experimental data is obtained.