Transcranial direct-current stimulation (tDCS) has been widely studied with theaim of enhancing local synaptic efficacy and modulating the electrical activity ofthe cortex in patients with neurological disorders.  The purpose of the present study was to determine the effect of a single sessionof tDCS regarding immediate changes in spatiotemporal gait and oscillations of thecenter of pressure (30 seconds) in children with cerebral palsy (CP). A randomized controlled trial with a blinded evaluator was conducted involving 20children with CP between six and ten years of age. Gait and balance were evaluatedthree times: Evaluation 1 (before the stimulation), Evaluation 2 (immediatelyafter stimulation), and Evaluation 3 (20 minutes after the stimulation). Theprotocol consisted of a 20-minute session of tDCS applied to the primary motorcortex at an intensity of 1 mA. The participants were randomly allocated to twogroups: experimental group - anodal stimulation of the primary motor cortex; andcontrol group - placebo transcranial stimulation.  Significant reductions were found in the experimental group regardingoscillations during standing in the anteroposterior and mediolateral directionswith eyes open and eyes closed in comparison with the control group (p<0.05).In the intra-group analysis, the experimental group exhibited significantimprovements in gait velocity, cadence, and oscillation in the center of pressureduring standing (p<0.05). No significant differences were found in the controlgroup among the different evaluations.  A single session of tDCS applied to the primary motor cortex promotes positivechanges in static balance and gait velocity in children with cerebral palsy. The electrical current flows from the electrodes and penetrates the skull,reaching the cerebral cortex. Although most of the current is dissipated among theoverlying tissues, a sufficient amount of current reaches the structures of the cortex,modifying the membrane potential of the cells and modulating cortex activityThere has been an increase in the number of studies stating that tDCS applied to themotor cortex can be used for the treatment of neurological disorders in children, suchas cerebral palsy (CP),Ninety percent of children with CP exhibit impaired gait due to diminished cortexexcitability, excessive muscle weakness, abnormal joint kinematics, and diminishedpostural reactionsKaski et al.The aim of the present study was to determine the effect of a single session of tDCSapplied to the primary motor cortex regarding immediate changes in spatiotemporal gaitand oscillations of the CoP during standing in children with CP classified at levels Ito III of the Gross Motor Function Classification System (GMFCS).Universidade Nove de Julho (UNINOVE), São Paulo, SP,Brazil, under process number 69803/2012. This study is registered with the BrazilianRegistry of Clinical Trials (process RBR-9B5DH7). All guardians signed a statement ofinformed consent agreeing to the participation of their children.The present randomized, sham-controlled, cross-sectional study  was carr,Children with a diagnosis of spastic CP were recruited from specialized clinics. Theinclusion criteria were classification at levels I, II and III of the GMFCSThe participants were randomized into the experimental and control groups based on theorder of inclusion into the study. A randomization list was generated using blocks ofsix  and four to minimize therisk of imbalance in the size of the groups.The procedures were carried out in a single day. Following Evaluation 1 , the children received 20 minutes of either active or sham (control group) tDCS. The children received tDCS at restand seated comfortably. A responsible therapist accompanied the stimulation session.Evaluation 2  was performed immediatelyfollowing tDCS and Evaluation 3 (twenty minutes after stimulation) was performed after20 minutes of rest. Three researchers carried out the procedures - two performed theevaluations and one performed the tDCS. The evaluations and tDCS were carried out inseparate rooms to ensure the blinding of the examiners. Only the researcher in charge ofthe application of the tDCS was aware of the allocation of the children to theexperimental and control groups.2 and moistened with saline solution. Thechildren in the experimental group received anodal stimulation of the primary motorcortex and those in the control group received placebo transcranial stimulation. Theanode was positioned over the primary motor cortex of the dominant hemispherefollowing the 10-20 international system of electrode placement forelectroencephalographytDCS is the application of a low-intensity direct current on the scalp using twoelectrodes (anode and cathode). A sufficient amount of current penetrates theoverlying tissues and reaches the structures of the motor cortex, modifying theneuronal membrane potential. Anodal stimulation enhances cortex excitability. ThetDCS device  included two non-metallic sponge surfaceelectrodes measuring 5 × 5 cm(r)system  with eight infrared cameras, the SMART-D INTEGRATEDWORKSTATION(r) with 32 analog channels and a synchronized video system.After the determination of the anthropometric measures , passive markers were placed at specificreference points directly on the skin for the evaluation of each segment of the body.The markers were placed over C7 and the sacrum as well as bilaterally over theacromion, anterosuperior iliac spine, greater trochanter, femoral epicondyle, femoralwand, tibial head, tibial wand, lateral malleolus, lateral aspect of the foot at thefifth metatarsal head and at the heel (only for static offset measurements), asdescribed by Davis et al.The evaluation of spatiotemporal gait variables  was performed using the SMART-D 140velocity (m/s): mean velocity of progression;cadence: number of steps in a time unit (steps/min); stride length (m): longitudinal distance between successive points of heelcontact of the same foot;step length (m): longitudinal distance between the point of initial contactof one foot and the point of initial contact of the contralateral foot;step width (m): distance between the rear end of the right and left heelcenterlines along the mediolateral axis;stance phase: % of gait cycle that begins with initial contact and ends attoe-off of the same limb.Mean and standard deviation values of gait velocity, cadence, step length, stridelength, and step width were used for the statistical analysis.(r). The child was instructed to remainin a quiet standing position on the platform, barefoot, arms alongside the body, gazefixed on a point marked at a distance of one meter at the height of the glabellum,with heels aligned and an unrestricted foot base. The children classified at levelIII of the GMFCS,Static balance was evaluated with the use of a force plate (Kistler model 9286BA),which allows stabilometric analysis through readings of oscillations of the CoP. Theacquisition frequency was 50 Hz, captured by four piezoelectric sensors positioned atthe extremities of the platform, which measured 40 × 60 cm. The data were recordedand interpreted using the SWAY software program (BTS Engineering) integrated to andsynchronized with the SMART-D 140Readings of displacement from the CoP on the X (anteroposterior) and Y axes were performed under two conditions: eyes open and eyes closed. Threeacquisitions of 30 seconds were obtained for each condition and the average of theacquisitions was used in the statistical analysis. The outputs of the force platformallowed us to compute the CoP time series in the anteroposterior direction and themediolateral direction. The output of the platform was processed to computequantitative parameters in the time domain. The anteroposterior and mediolateralcoordinates of the CoP trajectory underwent post-acquisition filtering using alow-pass filter with a cut-off frequency of 10 Hz. In the analysis, we identified andcomputed the range of CoP displacement in the anteroposterior direction (RANGEAPindex) and the mediolateral direction (RANGEML index), expressed in mmThe Kolmogorov-Smirnov test was used to determine the adherence of the data to theGaussian curve. Parametric distribution was demonstrated, the data were expressed asmean and standard deviation values. To verify the effect of transcranial stimulation(active and placebo) over the three Evaluations in each group, intragroup analysiswas performed. Intergroup analysis was performed to verify a possible effect obtainedby the experimental group (active stimulation). With these goals, two-way analysis ofvariance (ANOVA) was used with the Bonferroni post hoc test, considering thevariables: anteroposterior oscillations (open and closed eyes), mediolateraloscillations (open and closed eyes), and spatiotemporal gait parameters . The level ofsignificance was set to 0.05. The data was tabulated and processed using StatisticalPackage for the Social Sciences .Twenty children with CP were randomly allocated to the experimental group (active tDCSapplied to the primary motor cortex) or control group (sham tDCS). No statisticallysignificant differences between groups were found regarding the baseline data . ,-tDCS currently occupies an important place in studies addressing neuromotorrehabilitation due to its potential in optimizing the results of physical therapy,,Three-dimensional gait analysisThe present study offers important findings. The experimental group exhibited anincrease in gait velocity immediately following tDCS, but this increase was notmaintained for more than 20 minutes after the end of the stimulation. Although this wasa cross-sectional study involving a single session of tDCS, the results suggest that themomentary increase in cortex activation may have exerted an influence on motor controland gait. As the primary motor cortex was only stimulated for 20 minutes during rest,the authors did not expect the changes to be maintained in medium or long term. However,the findings could encourage future studies to combine tDCS with motor rehabilitationtherapies to determine whether this technique can assist in improving gait and posturalcontrol in children with CP.Gait velocity has an important relationship with the cadence. However, in this studythere was an increase in walking velocity without increasing cadence. The authorsbelieve that this fact can be explained by an increased step length.Analyzing a population of elderly individuals (n=9) with leukoaraiosis, an ischemiclesion of the cerebral white matter that results in gait and balance disorders, Kaski etal.,,Balance deficit resulting in frequent falls is one of the most limiting aspects ofCPVisual compensation is an important aspect of postural stability. In children with CP,oscillations are greater with eyes closed due to the lack of visual compensation. Theresults suggest that there was a momentary improvement in postural stability. Althoughthere are no studies that address the effects of tDCS on static balance, the authorsbelieve that greater effectiveness of the proprioceptive system may have resulted fromthe stimulation of the cortical area. Thus, the motor responses were effective inminimizing the oscillations with visual restriction. Similar results on the effect sizeof the oscillations of the CoP, with and without visual restriction, are observed onlywith more dynamic interventions, such as the use of ankle-foot orthoses,All clinical effects observed following the application of tDCS are directly related tocortex modulations resulting from stimulation dependent on the polarity of the current.Anodal stimulation increases cortex excitability, favoring the depolarization of theneuronal membrane, whereas the cathode has an inhibitory effect through thehyperpolarization of the neuronal membraneBased on the present findings, a single session of tDCS applied to the primary motorcortex in children with CP was capable of causing significant reduction inanteroposterior oscillation with eyes open and eyes closed and in mediolateraloscillation with eyes closed in comparison with the control group (tDCS sham). Moreover,increases in gait velocity, step length, and stride length were also observed afterstimulation. However, the results were not maintained for more than 20 minutes after theend of stimulation.


The Sahara experienced several humid episodes during the late Quaternary, associated with the development of vast fluvial networks and enhanced freshwater delivery to the surrounding ocean margins. In particular, marine sediment records off Western Sahara indicate deposition of river-borne material at those times, implying sustained fluvial discharges along the West African margin. Today, however, no major river exists in this area; therefore, the origin of these sediments remains unclear. Here, using orbital radar satellite imagery, we present geomorphological data that reveal the existence of a large buried paleodrainage network on the Mauritanian coast. On the basis of evidence from the literature, we propose that reactivation of this major paleoriver during past humid periods contributed to the delivery of sediments to the Tropical Atlantic margin. This finding provides new insights for the interpretation of terrigenous sediment records off Western Africa, with important implications for our understanding of the paleohydrological history of the Sahara. Given the absence of a major river system in the Western Sahara, the source of late Quaternary fluvial sediments along the West African margin remains unclear. Here, the authors present geomorphological data that reveal the existence of a large buried paleodrainage network on the Mauritanian coast. Astronomically forced insolation changes have driven monsoon dynamics and the periodical onset of humid episodes in North Africa over the last few million years, resulting in the ‘greening' of the Sahara and savannah expansion throughout most of the desert at times34556789Off the Western African margin, a region that lies today under the main corridor of Saharan dust plumes, fluvial signals have been identified in deep-sea sediments dated from Marine Isotope stage 5 ref. 511. These s511Recently, a spectacular 400-km-long submarine channel system—the Cap Timiris Canyon—has been discovered on the western Sahara margin off Mauritania121516121516In this paper, based on remote-sensing data, we identify the presence of a large paleodrainage network on the arid Mauritanian coast, shallowly buried at present under aeolian sediments. Combined with other geomorpholgical and sedimentary lines of evidence available in the literature, our findings suggest that a major river system was indeed reactivated during some of the humid periods of the last 245 kyrs, thus likely contributing to the delivery of sediments to the Tropical Atlantic margin at those times.To provide further constraints on the link between past sediment discharges to the Tropical Atlantic margin and fluvial activity in Western Sahara, we have analysed the geomorphology of the Mauritanian coastal area using remote-sensing imagery. We have used the Phased Array type L-band Synthetic Aperture Radar (PALSAR), which is one of the Japanese Advanced Land Observing Satellite remote-sensing instruments. PALSAR is an active microwave sensor using L-band frequency to achieve day-and-night and all-weather land observation. The L-band (1.2 GHz) radar has the capability to penetrate metres of low electrical loss material such as aeolian sand19Our radar observations of the coastal Mauritania area  provide Previous studies, focused on the morphology and the seismic structures of the Cap Timiris Canyon, have suggested that it may have been active for at least the last 245 kyr ref.  during p34Investigation of sedimentary records from the Eastern Mediterranean Sea off the Nile River has allowed the identification of nine distinct sapropel layers during the last 245 kyr ref. , all occ511Overall, the identification of the paleodrainage system in the Arguin Bay area is very coherent with the hydrological landscape of Western Sahara, especially during the most recent AHPs. The testimony of a fluvial activity on the Mauritanian coast during recent humid periods provides the missing link between the development of lakes over Algeria and Mauritania5112, representing close to a quarter of the surface area of Libya.As demonstrated by the Shuttle Imaging Radar (SIR) missions in the early eighties, a radar wave can penetrate dry superficial sediment layers and thus reveal unknown paleohydrological and tectonic structures19293335How to cite this article: Skonieczny, C. et al. African Humid periods triggered the reactivation of a large river system in Western Sahara. Nat. Commun. 6:8751 doi: 10.1038/ncomms9751 (2015).


Introduction: Confinement of biomolecules in structured nanoporous materials offers several desirable features ranging from chemical and thermal stability, to resistance to degradation from the external environment. A new generation of mesoporous materials presents exciting new possibilities for the formulation and controlled release of biological agents. Such materials address niche applications in enteral and parenteral delivery of biologics, such as peptides, polypeptides, enzymes and proteins for use as therapeutics, imaging agents, biosensors, and adjuvants.Areas covered: Mesoporous silica Santa Barbara Amorphous-15 (SBA-15), with its unique, tunable pore diameter, and easily functionalized surface, provides a representative example of this new generation of materials. Here, we review recent advances in the design and synthesis of nanostructured mesoporous materials, focusing on SBA-15, and highlight opportunities for the delivery of biological agents to various organ and tissue compartments.Expert opinion: The SBA-15 platform provides a delivery carrier that is inherently separated from the active biologic due to distinct intra and extra-particle environments. This permits the SBA-15 platform to not require direct modification of the active biological therapeutic. Additionally, this makes the platform universal and allows for its application independent of the desired methods of discovery and development. The SBA-15 platform also directly addresses issues of targeted delivery and controlled release, although future challenges in the implementation of this platform reside in particle design, biocompatibility, and the tunability of the internal and external material properties. Examples illustrating the flexibility in the application of the SBA-15 platform are also discussed. The last few decades have brought significant advances in the design and synthesis of novel drug delivery systems A systematic trend in drug development during the last two decades is the shift towards biologics expert opinion, we focus on recent advances regarding a compelling mesoporous silica candidate that fits this niche, Santa Barbara Amorphous-15 (SBA-15) Recent studies 2. Before evaluating any novel nanomaterial candidate for drug delivery, it is important to first evaluate where current materials fall short. Key properties that are desired in a nanomaterial candidate include selective compartmental distribution in organs and tissues, particle size uniformity, selective affinity-based interactions, material stability and biodegradability, versatile surface chemistry and tunable particle geometry Due to shortcomings of some synthetic nanoparticles, many researchers are also investigating the use of nanoparticles synthesised of biological materials. These materials usually consist of DNA As described previously, it is difficult to tune the specific properties of many nanoparticles under development in a manner that facilitates the desired biological response. Changing one desired property might change another one that is equally desirable for an application 3. An ideal nanoporous material for drug delivery would address many of the challenges highlighted previously. Porous materials with biomolecules confined in the pores allow for separation of particle–biomolecule and particle–physiological interactions to the interior and exterior of a particle, respectively. The pore sizes that allow for the separation of these interactions for protein biologics are typically in the mesoporous range (2 – 50 nm pore diameter). Challenges of engineering the interface between the internal pore environment and the biomolecule include issues such as proteolysis, pH induced aggregation, shear induced denaturing, hydrophobic denaturing and the regulation of biomolecular diffusion As the use of porous materials isolates particle–therapeutic interactions to the interior of the particle, the extra particle challenges deal primarily with the physiological interactions of the particle and global targeting of the therapeutic. Extra particle challenges include targeted delivery of the therapeutic, isolation of particles, crossing of desired barriers and mitigation of non-discriminant interactions with proteins and biomolecules in serum and other biological fluids. It is essential for each of these challenges to be addressed to localise the therapeutic effect, minimise toxicity issues and achieve effective therapeutic delivery.There are several classes of porous nanoparticles currently being investigated to address challenges in therapeutic delivery. These include polymeric gels 4. 2) SBA-15 is a mesoporous silica in which the pores are templated by tri-block copolymer micelles 20PO70EO20, where EO is ethylene oxide and PO is propylene oxide) in an acidic aqueous environment. Acidic, high-ionic strength P123 micelles transform from spherical to cylindrical when tetraethyl orthosilicate is added to the solution SBA-15 is synthesised by first dissolving Pluronic 123 (P123) tri-block copolymer . The authors found that the model proteins adsorbed on the propylated surface were less catalytically active and had significant changes to their secondary structure when compared with the same proteins free in solution. The changes in secondary structure showed no dependence on the diameter of the pore. These results suggest that a phase inversion and denaturing occurs on a hydrophobic interface, as one might suspect As discussed previously, SBA-15 contains hexagonally ordered cylindrical mesopores with a narrow pore size distribution. Compared with most other mesoporous silicas, this gives it the unique ability to expose the biological cargo to only concave curvature upon adsorption. This property of SBA-15 was recently investigated by Sang et al. were observed as a corresponding increase in protein activity. Also using activity as a gauge for stability, Radhakrishna et al. In trying to understand why this effect is so dramatic, one can look at a structural analogue found in nature, the GroEL/ES chaperonin complex. The GroEL/ES chaperonin complex is required to finish the proper folding and for the refolding of some misfolded bacterial proteins et al. Although these results are promising, they merely imply that stabilisation effects are present. To further demonstrate the protection effects, Lynch et al. achieved nearly 100% platelet-like particles Apart from pore curvature, other material properties, such as the particle size, mesopore length and particle shape of SBA-15, can also be modulated or tuned to achieve the desired effect. For example, by changing the synthesis process, Zu et al. et al. in vitro cytotoxicity in the Caco-2 cell line. Similarly, Ahmadi et al. found that amino functionalisation of SBA-15 significantly increases loading and release duration of ibuprofen et al. found that high hydroxylation of the SBA-15 surface increases the already enhanced release of the poorly soluble therapeutic itraconazole into a simulated biological fluid in vivo models Another design parameter that allows for significant modulation of the particle properties is surface functionalisation. The functionalisation of silica surfaces is well established; this imparts flexibility and reliability on modifying the surface chemistry of SBA-15. In addition, due to the stability of silane bonds, after initial silica functionalisation, well-known organic methods can be used to complete the process using click chemistry et al. investigated the targeting of HeLa and A549 cancer cells mediated by the folic acid receptor Just as internal surface functionalisation of SBA-15 has been used to control the internal environment of SBA-15, external surface functionalisation is also used to directly address the extra-particle challenge of targeted delivery. This allows for the possibility of limiting therapeutic activity to specific areas and targets such as the blood–brain barrier et al. et al. have synthesised organic supramolecule megagates that cap the pores of SBA-15 until exposed to a pH below 5 Controlled release of therapeutic cargo can also be regulated by methods other than just the general surface properties, which can yield even greater design flexibility Although the insights and tools developed in each of these studies do not separately give way to the next super-drug delivery platform, the nature of the tools and the ability to integrate them leads to many design possibilities. Stability and protection effects from the physiological environment are rendered by the fundamental effects of confinement, curvature and pore surface properties. Targeted delivery and physiological interactions are addressed by flexible external surface functionalisation techniques that allow for a wide range of functional groups. Controlled release is realised by adaptive capping and functionalisation methods that are governed by processes ranging from simple conformational changes to elaborate supramolecular interactions. 6. Numerous tools are being developed for the delivery of biological therapeutics. However, many of these tools require the direct modification of the active biologic of interest et al. have been able to prevent gelation at very high protein concentrations through the use of trehalose as a crowding agent One of the prominent challenges in the delivery of biological therapeutics is that of protein crowding. The difficulties arise at high protein concentrations, for which, in addition to destabilising protein–protein interactions, therapeutic biologics can become too viscous to deliver via injection due to gelation et al. evaluated the biocompatibility of mesoporous silicas with particle sizes of 150 nm, 800 nm and 4 µm  with pore sizes of 3, 7 and 16 nm, respectively. Subcutaneous injection of the materials were well tolerated in a rat model and resulted in elimination of the material progressively over 3 months while showing good biocompatibility on histology. However, the silicates were also shown to have a significant degree of toxicity to mesothelial cells and resulted in death or euthanasia when administered via intraperitoneal or intravenous injection to SV129 mice. On the other hand, Lu et al., using a nude mice model, showed that fluorescent mesoporous silica nanoparticles of 130 nm with 2 nm pores are well tolerated via intraperitoneal and intravenous administration et al. have also shown that almost all of the injected particles are excreted via urine and feces within 4 days of administration. Huang et al. conducted an in vivo study on the effect of mesoporous silica nanoparticles on nude mice xenografted with human malignant melanoma cells (A375) in vitro studies confirmed that the nanoparticles promoted cellular proliferation and accelerated cell cycle progression of the tumour cells. Furthermore, Huang et al. determined that these effects are due to a decreasing of endogenous reactive oxygen species. These three seemingly conflicting studies emphasise contradictory views on the biocompatibility of these materials, and more importantly the fact that the flexibility of the SBA-15 platform is essential for its successful implementation. Size, shape, porosity and surface chemistry are all important, and the ability to control them in SBA-15 is crucial for successful biocompatible design using this platform. This flexibility in design allows for the accurate tuning of many material properties, including surface functionalisation. The difficulty with most current methods is that they impact the functionalisation of the whole, internal and external, surface of SBA-15 particles. This leaves gaps in the literature when selective functionalisation of the internal and external surface is desired. Although methods do exist As a consequence, the challenge in implementing the SBA-15 platform lies in the particle design. This revolves around particle biocompatibility and the tunability of both the internal and the external material properties. Although the biocompatibility of this platform is crucial for its success as a therapeutic delivery system, the topic is too broad to be thoroughly considered here. However, three studies are discussed to highlight the work in this area showing the challenges associated with various routes of administration, particle sizes and particle concentrations Addressing these challenges will enable the rational modulation of both the thermodynamic and kinetic aspects of therapeutic delivery. This can range from designing a strong thermodynamic driving force for protein desorption from the particle, while creating a kinetic barrier to prevent delivery until located at the targeted site (design 1), to optimising the internal pore structure for stability and activity, and the pore size for quick substrate diffusion (design 2).A possible application of the first hypothetical design would be the delivery of a biological therapeutic across the blood–brain barrier. This could be achieved by the selective functionalisation of the exterior surface of the particle with appropriate ligands and biocompatibility coating. In this case, the ligands could be a ‘marker of self’ peptide resembling CD47 In the second design, pore structure and size could be optimised to increase stabilisation of the protein cargo as well as the substrate diffusion rate to the catalytic protein therapeutic. This would increase the effectiveness of the protein used as the catalytic therapeutic. One application could be the confinement of lactase to address lactose intolerance. The stabilisation and protection effects due to confinement would protect the therapeutic when exposed to the extreme environments of the gastrointestinal tract. Particle size and external functionalisation could then prevent the particle from leaving the gastrointestinal tract and select for the desired residence time of the therapeutic particle. An overview of each of these designs can be seen in Each of these examples illustrates the variability of application without the requirement to modify the intended active therapeutic. This along with the inherent stability effects due to confinement and flexibility in design are the major strengths of the SBA-15 platform.Article highlights.Confinement of biological therapeutics in porous materials enables the inherently distinct intra and extra-particle environments to be leveraged during the therapeutic design process.SBA-15 allows independent tuning of key material parameters.The SBA-15 platform allows for the delivery of protein therapeutics without direct modification of the desired therapeutic.This box summarizes key points contained in the article.The authors gratefully acknowledge the support of the NSF through Grant No. 0333314 and via CBET-0967937, and the EPSRC through a Frontier Engineering Award for the Centre for Nature Inspired Engineering, EP/K038656/1. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.


Background: There is a growing concern, but limited evidence, that price promotions contribute to a poor diet and the social patterning of diet-related disease.Objective: We examined the following questions: 1) Are less-healthy foods more likely to be promoted than healthier foods? 2) Are consumers more responsive to promotions on less-healthy products? 3) Are there socioeconomic differences in food purchases in response to price promotions?Design: With the use of hierarchical regression, we analyzed data on purchases of 11,323 products within 135 food and beverage categories from 26,986 households in Great Britain during 2010. Major supermarkets operated the same price promotions in all branches. The number of stores that offered price promotions on each product for each week was used to measure the frequency of price promotions. We assessed the healthiness of each product by using a nutrient profiling (NP) model.Results: A total of 6788 products (60%) were in healthier categories and 4535 products (40%) were in less-healthy categories. There was no significant gap in the frequency of promotion by the healthiness of products neither within nor between categories. However, after we controlled for the reference price, price discount rate, and brand-specific effects, the sales uplift arising from price promotions was larger in less-healthy than in healthier categories; a 1-SD point increase in the category mean NP score, implying the category becomes less healthy, was associated with an additional 7.7–percentage point increase in sales . The magnitude of the sales uplift from promotions was larger for higher–socioeconomic status (SES) groups than for lower ones . Finally, there was no significant SES gap in the absolute volume of purchases of less-healthy foods made on promotion.Conclusion: Attempts to limit promotions on less-healthy foods could improve the population diet but would be unlikely to reduce health inequalities arising from poorer diets in low-socioeconomic groups. Price promotions are commonly used in store with the aims of boosting purchasing by reducing the price of products as well as possibly stimulating impulsive purchases by increasing the prominence of items in store . There is a growing concern that such promotional activities by the food industry may contribute to poor dietary intake particularly in individuals who are more socially deprived –3. It waHowever, there is a paucity of empirical evidence available in the public domain, and the existing claims about a bias in the use of price promotions toward less-healthy items largely rest on anecdotal reports. Although the general responsiveness of consumers to price promotions received substantial attention in the marketing literature , 6, and We also sought to explore whether social disparities in the healthiness of food purchased were attributable to differences in responses to retail price promotions. In an earlier study, by using the same survey data, we showed significant socioeconomic patterning in the healthiness of food purchases . However1) Are less-healthy foods more likely to be promoted than healthier foods? 2) Are consumers more responsive to promotions on less-healthy products than promotions on healthier ones? 3) Are there socioeconomic differences in food purchases in response to price promotions?We designed our study to address the following 3 questions: We used a secondary data source, the Kantar WorldPanel survey , 16, whiThe survey includes purchase records of all foods and beverages that were taken home from supermarkets and similar stores in 2010. Sampled households were asked by the data company to record all purchases using barcode scanners and to send digital images of cash-register receipts to the company. The data contain rich information on purchases, including the price at which they were bought, whether they were bought on promotion, the number of packets purchased, and the retail chain from which the product (Stock Keeping Unit) was purchased. The data also include detailed information on product characteristics, including information on the brand, manufacturing company, and nutritional content.Supplemental Table 1 for more information). With the use of transaction records in the data, we calculated the total number of units of each product sold to panel households across the country over 52 wk. In keeping with common practice in the related literature , a price promotion is recorded for a given item in a given store only if that item has been purchased on promotion from that store by panel households. This purchase-based nature of existing data sets has thus far prevented researchers from measuring the frequency of price promotions at population level.In the absence of a directly observable measure in the data, we estimated the frequency of promotions for each product by exploiting a particular feature of the retail policy in major United Kingdom grocery retailers. We focused on the following 11 “main parties” of United Kingdom multiple grocers: Tesco , Asda , Sainsbury , Morrisons , Waitrose , Iceland , Lidl , Aldi , M&S , Netto , and Budgens (market share information not available), as defined by the United Kingdom Competition Commission . The totSupplemental Data section 2 for additional details.The frequency of promotions for each product was defined by the number of branches  that ranThe nutrient profiling (NP) model developed by the United Kingdom Food Standards Agency was used to capture the healthiness of products . This mej in category c, the following base model was specified:To address this question, we set up a product-level regression model of sales and assessed the relation between the frequency of promotions and NP score. In supermarkets, each product was nested by product category (135 categories in our data), and hence, our product-level data set had a 2-level structure . First, we estimated the association between the frequency of promotions and NP score of various food categories . Next, we estimated the relation between promotions and the NP score at the product-level separately by the food category . These 2 step estimations were conducted simultaneously via a hierarchical regression approach . For itejc refers to the frequency of promotion of item j in category c and NP represents the nutrient profile. The term ejc is the idiosyncratic error. This basic estimation was used to tell whether less-healthy items were more frequently promoted than healthier ones. We further specified that the baseline frequency of promotions (intercept: β0c) and the association with the NP score (slope: β1c) varied by dietary category, and this variation was a function of the genuine healthiness of each dietary categoryFoPc. Now, the model could distinguish between product-level  effects and category-level  effects of healthiness, which were estimated separately in the regression analysis. See Supplemental Data section 3 for full technical details.To address this question, we investigated differential effects of the frequency of promotions on product sales by the NP score of products. The analysis assessed whether price promotions increased sales of less-healthy compared with healthier foods (between-category effect). The analysis also addressed whether sales of less-healthy versions within a given food category increased more in response to promotions than did healthier versions in the same food category (within-category effect). Again a similar hierarchical regression approach was used. The baseline product-level purchases equation is given byj in category c that were purchased by the panel households over 52 wk. The interaction term [log(FoPjc)×NPjc] was used to measure whether and, if so, to what extent the effect of promotions varied by the healthiness of the product. The vector Zjc included a set of product-level covariates known to affect sales, including the reference price, average rate of price discount when promoted, and a set of indicators of brands (which captured the brand-specific features of each product).The outcome variable was the log of total number of products Similar to the previous analysis, category-specific coefficients were modeled as follows:The model nested the within-category and between-category sales effects of promotion by healthiness, which, again, were estimated separately in the regression analysis. All models were estimated via a restricted maximum likelihood technique . See Sup1) high–socioeconomic status (SES) households, 2) middle-SES households, and 3) low-SES households and repeated the previous analysis (on the basis of NP scores) for each group. The SES of the household was defined by the occupation of the household head using the United Kingdom Registrar General’s classification  rather These SES differences in the responsiveness to promotions were more pronounced in healthier than in less-healthy categories . Table 2There was also an SES gap in the sales of both healthier and less-healthy foods that were made off promotion . This reTo our knowledge, the current study provides the first population-level quantitative assessment of the relation between the frequency of price promotions and healthiness of food purchases in the main supermarket chains in the United Kingdom. The analysis involved a considerably larger sample size than in existing studies on price promotions. Our study focused on temporary price changes that often augment the prominence of items in the store through tags and placement. Hence, the research usefully complemented existing studies on the role of price in healthy food purchasing more generally, which have had implications mainly for taxation or subsidization .To our knowledge, we also provided the first systematic assessment of a channel through which social disparity of food purchases and intake may occur. Although we and others previously showed social patterning of diet quality  , 32–34, In interpreting the findings, several limitations need to be borne in mind. First, our measure of the frequency of promotions was inevitably limited. The construction of the variable relied on the national pricing policy operated in leading United Kingdom supermarkets. However, the policy is known to be imperfectly adhered to in places characterized by a highly competitive market, such as in central London . MoreoveSupplemental Table 12). Moreover, we did not address potential differences in purchasing across social groups within a given store.Second, the current study highlighted differential responses to price promotions by social groups, with the assumption that different social groups were exposed to the same promotional environment at the national level. However, United Kingdom supermarket chains have different main target consumers and operate in different parts of the country, and hence, the promotional environment may be segmented by social groups. Our sensitivity analyses that looked at shoppers’ exposure to promotions  showed similar socioeconomic patterning in responses to promotions , thereby excluding relatively smaller grocery chains and privately owned stores. Purchasing patterns of consumers as well as marketing strategies in those stores may have been different from that of the main parties. Therefore, our findings may not be entirely generalizable.Our measure of the frequency of price promotions provided an indication of variability of price promotions for individual products at the national level, but more-refined ways to measure the frequency of price promotions  should be developed in future research . MoreoveIn the current study, we examined overall differences in responses to price promotions by SES groups at the population level. Future research could investigate the issue at the store level so that consumers’ responses to price promotions are analyzed within the same marketing strategy and variety of food products.Detailed analysis of various types of price promotion  could be valuable. Recent evidence showed that restricting multibuys has failed to change the overall volume of alcohol purchased . HoweverFinally, the effects of price promotion on the whole food basket purchased  should be evaluated. A sizeable proportion of total purchasing involved foods on promotion , which mOur findings suggest that policies that restrict price promotions on less-healthy food categories could help achieve healthier nutrient profiles of shopping baskets for the population on average, which is likely to lead to improvements in the nutritional value of food consumed. However, we did not find evidence that restricting promotions on less-healthy versions of products within a given category would achieve a similar benefit.In conclusion, our results imply an intriguing effect in relation to socioeconomic inequality. The SES difference in the responsiveness to promotions was more marked in healthier rather than less-healthy food categories. Moreover, the SES gap in the sales of less-healthy foods was predominantly driven by differences in off-promotion sales. Hence, the restriction of price promotions on less-healthy food categories would be unlikely to reduce the SES gap in the healthiness of food purchasing. The quest continues for measures to improve diet quality for the population as a whole while simultaneously decreasing health inequalities.


Shortly after implantation the embryonic lineage transforms from a coherent ball of cells into polarized cup shaped epithelium. Recently we elucidated a previously unknown apoptosis-independent morphogenic event that reorganizes the pluripotent lineage. Polarization cues from the surrounding basement membrane rearrange the epiblast into a polarized rosette-like structure, where subsequently a central lumen is established. Thus, we provided a new model revising the current concept of apoptosis-dependent epiblast morphogenesis. Cell death however has to be tightly regulated during embryogenesis to ensure developmental success. Here, we follow the stages of early mouse development and take a glimpse at the critical signaling and morphogenic events that determine cells destiny and reshape the embryonic lineage. Sperm entry triggers the “big bang” that transforms a fertilized egg into a new organism. Cell fate choices generate a diversity of tissues that undergo morphogenetic transformations, reshaping the developing embryo. The first two cell fate decisions set up the embryonic and extraembryonic lineages. The embryonic lineage or the epiblast (EPI) contains pluripotent progenitors that give rise to all tissues of the foetus. The signals that organise and support the development of the EPI are provided by derivatives of the two extraembryonic lineages - the trophectoderm (TE) and the primitive endoderm (PE) A. The fiMultiple cell generations maintain and expand the embryonic and extraembryonic structures throughout embryogenesis. This continuity relies on the tightly balanced processes of cell death and survival. Cell death can occur following the paths of necrosis or apoptosis. Necrosis is not a regulated process and is usually a result of physical damage or ischemia that compromises cell integrity leading to the release of cell contents into the external environment. In contrast, programmed cell death (PCD) or apoptosis is a multilevel, heavily regulated process, activated by external or cell intrinsic stimuli. The intrinsic apoptotic program can be triggered as a response to DNA damage, high calcium and oxidant levels or lack of survival signals, whereas the extrinsic apoptotic cascade is downstream of ligand activated cell surface receptors. Membrane blebbing, nuclear condensation and formation of apoptotic bodies are the typical morphological characteristic of cells undergoing apoptosis. Subsequently, the dying cells and debris are rapidly cleared from the tissue by efferocytosis ,8.Apoptosis is suggested as a default cell destiny that has to be continuously suppressed by survival signals . Such siThe observed incidence of apoptosis at the blastocyst stage differs between the TE and the inner cell mass (ICM). PCD in the TE is a relatively rare event, whereas the rates of apoptosis in the ICM are significantly higher ,21. It iAfter the second lineage segregation is complete, the mature blastocyst hatches out of the zona and initiates implantation. The mural TE mediates the first interactions of the implanting blastocyst with the maternal environment. The direct contact between the TE cells and the uterine epithelium induces apoptosis at the attachment site, allowing penetration of the embryo into the underlying stroma. This apoptotic process is suggested to be a result of TNF-receptor I activation that triggers Caspase-3 mediated local PCD of the luminal epithelium ,29.As the embryo invades the maternal environment the surrounding stroma proliferates and transforms into the decidua that supports the growth of the developing egg cylinder and ensures foetomaternal immune tolerance . FollowiFollowing implantation the polar TE forms the extraembryonic ectoderm (ExE) at the proximal region of the egg cylinder. The ExE contains the multipotent progenitors of the trophoblast lineage that form the embryonic portion of the placenta. The PE layer differentiates into parietal endoderm (PE) that migrates over the mural TE surface and visceral endoderm (VE) that engulfs the developing egg cylinder. Signalling centers of the VE pattern the underlying EPI, breaking its symmetry to establish the anterior – posterior axis –43.The process of EPI re-organization during peri-implantation stages has been a long-standing mystery. Embryos at those stages are no longer free-floating and as they invade the maternal tissues they become relatively inaccessible. The time of implantation is one of the most critical periods of development. Only embryos that successfully attach and implant stand a chance of completing embryogenesis. The process of implantation also boosts cell proliferation and, for the first time, embryo growth is initiated. As the egg cylinder emerges the EPI dramatically changes its morphology from a simple ball of unpolarised cells into a cup-shaped pseudostratified epithelium, surrounding the proamniotic cavity.Cavitation and hollowing are the two major paths through which a solid cohort of cells can be transformed into a tube by the generation of a central luminal space. Cavitation is an apoptosis-dependent mechanism of cell elimination in the core of a coherent mass of cells. Hollowing is apoptosis-independent, but requires separation of apical membranes in a radially polarized structure ,5. In viAccording to a long-standing simple and elegant two-step model, the EPI was thought to be reshaped by an apoptosis driven process of cavitation A. This mUsing embryos directly isolated from the uterus or blastocysts cultured in vitro throughout the corresponding peri-implantation stages we revealed a strikingly different sequence of events B. The poThe BM function in the establishment of apical basal polarity can be substituted in vitro by culturing isolated ICMs in 3D ECM. The EPI cells polarize and initiate lumenogenesis in the absence of a surrounding VE layer, indicating that a death signal from the VE is not required for this process. Similar morphogenetic changes can be induced in ES cells when they are embedded into 3D ECM at low density. The ES cell spheres formed in these conditions efficiently polarize and establish central lumen, mimicking the pre- to post-implantation transition of the EPI lineage . TogetheCytoplasmic movements in the zygote and cell division patterns of the 2-cell stage embryo are the earliest indicators of an embryo's potential for full development to term. As cleavage divisions progressively generate smaller blastomeres, multiple paracrine and autocrine signals promote embryo viability. The balance between cell survival and apoptosis is tightly regulated to maintain the functional integrity of the early lineages. A burst of cell proliferation in the implanting embryo expands all cell lineages, alongside a dramatic morphogenetic transformation of the EPI. Cells that are damaged or miss-positioned are most likely eliminated through apoptosis; however the process of PCD is dispensable for the morphogenetic changes that take place at that time. The BM components secreted by the extraembryonic lineages establish a niche that provides polarization cues to the pluripotent cells of the implanting embryo. In line with this, embryos that fail to assemble BM or lack β1-integrin receptors die during the implantation stages ,49,50. S2+ oscillations that induce contractions of the actomyosin network, manifested as rhythmic cytoplasmic movements. Using high-speed time-lapse imaging the patterns of cytoplasmic movements have recently been related to the developmental potential of the zygote. Frequent speed peaks, as well as a low mean basal speed of cytoplasmic movement, were found to be associated with a low rate of development to the blastocyst stage in vitro and to birth in vivo (PCD is involved in the process of oogenesis, where most of the oocytes are lost during the first stages of meiotic prophase I . Thus, t in vivo B. Thus, The regulative development of pre-implantation embryos enables adaptation to reductions in cell number. If one of the 2-cell stage blastomeres is destroyed, an individual totipotent blastomere can still complete embryonic development . Within


Human speech consists of a variety of articulated sounds that vary dynamically in spectral composition. We investigated the neural activity associated with the perception of two types of speech segments: (a) the period of rapid spectral transition occurring at the beginning of a stop-consonant vowel (CV) syllable and (b) the subsequent spectral steady-state period occurring during the vowel segment of the syllable. Functional magnetic resonance imaging (fMRI) was recorded while subjects listened to series of synthesized CV syllables and non-phonemic control sounds. Adaptation to specific sound features was measured by varying either the transition or steady-state periods of the synthesized sounds. Two spatially distinct brain areas in the superior temporal cortex were found that were sensitive to either the type of adaptation or the type of stimulus. In a relatively large section of the bilateral dorsal superior temporal gyrus (STG), activity varied as a function of adaptation type regardless of whether the stimuli were phonemic or non-phonemic. Immediately adjacent to this region in a more limited area of the ventral STG, increased activity was observed for phonemic trials compared to non-phonemic trials, however, no adaptation effects were found. In addition, a third area in the bilateral medial superior temporal plane showed increased activity to non-phonemic compared to phonemic sounds. The results suggest a multi-stage hierarchical stream for speech sound processing extending ventrolaterally from the superior temporal plane to the superior temporal sulcus. At successive stages in this hierarchy, neurons code for increasingly more complex spectrotemporal features. At the same time, these representations become more abstracted from the original acoustic form of the sound. During the articulation of speech, vibrations of the vocal cords create discrete bands of high acoustic energy called formants that correspond to the resonant frequencies of the vocal tract. Identifying phonemic information from a speech stream depends on both the steady-state spectral content of the sound, particularly the relative frequencies of the formants, and the temporal content, corresponding to fast changes in the formants over time. Speech sounds can be divided into two general categories, vowels and consonants, depending on whether the vocal tract is open or obstructed during articulation. Because of this difference in production, vowels, and consonants have systematic differences in acoustic features. Vowels, which are produced with an open vocal tract, generally consist of sustained periods of sound with relatively little variation in frequency. Consonants, on the other hand, are voiced with an obstructed vocal tract, which tends to create abrupt changes in the formant frequencies. For this reason, vowel identification relies more heavily on the steady-state spectral features of the sound and consonant identification relies more on the momentary temporal features Kent, .Research in animals suggests that the majority of neurons in auditory cortex encode information about both spectral and temporal properties of sounds  was used to investigate the cortical organization of phonetic feature encoding in the human brain. A main question is whether there are spatially distinct parts of auditory cortex that encode information about spectrally steady-state and dynamic sound features. Isolating feature-specific neural activity is often a problem in fMRI because different features of a stimulus may be encoded by highly overlapping sets of neurons, which could potentially result in similar patterns and levels of BOLD activation during experimental manipulations. One way to improve the sensitivity of fMRI to feature-specific encoding is to use stimulus adaptation . All subjects were right-handed, native English speakers, and had normal hearing based on self report. Subjects gave informed consent under a protocol approved by the Institutional Review Board of the Medical College of Wisconsin.http://www.fon.hum.uva.nl/praat). The acoustic parameters for the synthesizer were derived from a library of spoken CV syllables based on a male voice  were asked to identify each stimulus as one of the 12 possible CV syllables, as a different CV syllable, or as a non-speech sound. Based on the pilot study results, several of the Non-Phonemic and Single-Formant stimuli were removed from the stimulus set because they sounded too speech-like, and several of the Phonemic stimuli were removed because they were too often misidentified for another syllable or non-speech sound. A final stimulus set was chosen that consisted of Phonemic, Non-Phonemic, and Single-Formant versions of the syllables: /ba/, /bi/, /bae/, /ga/, /gi/, /gae/. In the final set, the Phonemic, Non-Phonemic, and Single-Formant stimuli were identified by participants of the pilot study as the original syllable (from which the syllable was derived and re-synthesized) at an average accuracy of 90, 46, and 13%, respectively.The stimuli were synthesized speech sounds created using the KlattGrid synthesizer in Praat  and five functional scans . Functional scans were collected using a sparse-sampling procedure in which stimuli were presented during a silent period between MR image collection  of trials were missing either one or two of the six stimuli. To ensure that subjects were attending to the stimuli during the experiment, subjects were required to hit a button when they detected a missing stimulus. Compliance with the task was assessed, but image data from the trials with missing stimuli were excluded from the analysis. Within each run 8 trials were presented per condition producing a total of 80 trials per condition across both sessions. An additional 8 trials of rest  were included in each run. Trials were presented in blocks containing 4 trials of the same condition. The order of the blocks was randomized across runs and across participants.The experiment was organized in a 3 × 4 factorial design with the three stimulus types  presented in four different adaptation configurations  resulting in a total of 12 conditions. The conditions were presented in trials consisting of six stimuli presented every 380 ms followed by a single MR volume acquisition lasting 1.8 s. A small percentage . Additional protective ear muffs were placed over the headphones to attenuate scanner noise.3) into the atlas space and spatially filtered using a Gaussian window (FWHM = 5 mm). Our primary research questions were focused on differences in activation in auditory areas, therefore, we confined our analysis to a set of voxels that included the entire superior, middle, and inferior temporal lobe and extending into the inferior parietal and lateral occipital lobes.The fMRI data were analyzed using AFNI  was performed on one of the contrasts. Mean effect sizes for each cluster were calculated by dividing the amplitude of the contrast values by the mean signal level and then taking a mean across all the voxels in the cluster. The maps are displayed on an inflated surface brain of the ANTS-derived atlas created using Freesurfer  estimate of temporal auto-correlation. Contrasts between conditions were evaluated at the group level using a mixed-effects model. To correct for increased type 1 error due to multiple comparisons, the voxels in the resulting statistical maps were initially thresholded at Figure 4), which we refer to as ventral STG, and distinguish this area from the more superior side of the STG, which we refer to as dorsal STG. There was less activity during Phonemic trials compared to Single-Formant trials in both hemispheres in the superior temporal plane (STP), specifically the medial portion, and in the posterior part of the middle temporal sulcus. Less activity during Phonemic compared to Non-Phonemic trials was found in a smaller cluster in the planum polare in the right hemisphere. Single-Formant trials had greater activity than Non-Phonemic trials in the left planum temporale.Differences in BOLD activation between the three stimulus types are shown in Figure To test for adaptation effects, each of the three adaptation conditions  were compared to the Baseline adaptation condition, in which all six stimuli in the trial were identical. Each of the adaptation contrasts included all three stimulus types. The resulting maps are shown in Figure p < 0.05 with the same corrected alpha level of 0.05  and the three adaptation contrasts  Figure . Voxels In the sections of cortex in the dorsal and ventral STG that showed activation in the stimulus and adaptation contrasts, we did not find significant interactions between adaptation and stimulus type. However, significant interaction effects were seen in several clusters outside of this region  were larger than those for the transition condition (T). Second, direct contrasts between the T and SS conditions Figure  showed gGreater activation for the T condition was found in several areas outside of auditory cortex. It has been suggested that vowels and consonants contribute differently to speech perception, with vowels containing the majority of acoustic information about prosody and segmentation, and consonants providing linguistic-based information about lexical identity (Nespor et al., Higher levels of activity in the bilateral ventral STG were seen for the Phonemic condition compared to the Non-Phonemic and Single-Formant sounds. This is consistent with findings from a large body of studies that have found greater activation in this area in response to speech syllables compared to non-speech auditory controls (Obleser et al., The results from the current study support the view that there are multiple hierarchical processing streams extending from primary auditory cortex to anterior, posterior, and lateral parts of the temporal lobe (Rauschecker et al., Finally, on the medial side of the STP, a larger response was found for Non-Phonemic and Single-Formant sounds compared to Phonemic sounds. This area did not activate in the adaptation contrasts. Other studies have observed a similar preference for non-speech over speech sounds in this region (Tremblay et al., In conclusion, we identified distinct regions of auditory cortex that were differentially sensitive to acoustic form and stimulus type, suggesting a hierarchical organization of auditory fields extending ventrolaterally from primary auditory cortex to the STS and with varying sensitivity to acoustic form along the anterior to posterior axis of the STG. These results extend our understanding of the brain areas involved in auditory object identification and speech perception.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The evidence for the slow development from gathering and cultivation of wild species to the use of domesticates in the Near East, deriving from a number of Epipalaeolithic and aceramic Neolithic sites with short occupational stratigraphies, cannot explain the reasons for the protracted development of agriculture in the Fertile Crescent. The botanical and faunal remains from the long stratigraphic sequence of Chogha Golan, indicate local changes in environmental conditions and subsistence practices that characterize a site-specific pathway into emerging agriculture. Our multidisciplinary approach demonstrates a long-term subsistence strategy of several hundred years on wild cereals and pulses as well as on hunting a variety of faunal species that were based on relatively favorable and stable environmental conditions. Fluctuations in the availability of resources after around 10.200 cal BP may have been caused by small-scale climatic fluctuations. The temporary depletion of resources was managed through a shift to other species which required minor technological changes to make these resources accessible and by intensification of barley cultivation which approached its domestication. After roughly 200 years, emmer domestication is apparent, accompanied by higher contribution of cattle in the diet, suggesting long-term intensification of resource management. Explaining the Beginnings of Agriculture: An Accumulated Record. Since the first half of the 20th century the importance of agriculture in the history of humankind has been continuously emphasized along with a steady development of models on the natural conditions and cognitive circumstances under which the neolithization process took place.From a bioarchaeological perspective many questions on the associated changes seem to be well understood. During the last decades identification criteria for distinguishing wild and domesticated plant species have improved (e.g. ). In somClimatic fluctuations following the end of the last glacial maximum have been comprehensively investigated and favored by some to represent a key catalyst for the development of agriculture under a combined contribution of changes in resource availability and demographic pressures –22. Othe Homo sapiens evolution, the form and functionality of the human brain have often been considered to have changed little, although the cognitive and cultural faculties as tangible through the archaeological record have changed considerably over time .This paper focuses specifically on two aspects of the archaeological record that are particularly relevant to the investigation of agricultural origins: archaeobotanical and zooarchaeological assemblages. Our goal is to address the major epistemological issue of building broad-scale, cross-regional generalizations on the basis of a fragmentary record.An impediment to the investigation of the local, long-term transitional developments from cultivating wild and later domesticated species, is the generally short-term occupation at aceramic Neolithic sites, in most cases spanning less than 2.000 years. The nature of the evidence on the beginnings of agriculture is therefore accumulated from disparate locations across the Fertile Crescent. Emerging problems from adding up snapshots from sites at different geographic locations with the goal of reconstructing a developmental sequence relate to the above described differences in local climatic and environmental conditions, as well as in socio-cultural identities, landscape perceptions and associated strategies of the prehistoric populations . ThThe long-term archaeobiological record from the aceramic Neolithic site of Chogha Golan, representing more than 2.000 years, allows the investigation of a multifactorial interplay of humans with their environment, including adaptive cycles at the transition to agriculture.Chogha Golan and Its Significance for Understanding the Neolithization Process. The aceramic tell site of Chogha Golan is situated at the lower ranges of the Central Zagros Mountains, about 30 km north of Mehran (Amirabad plain/Ilam Province/Iran) (ce/Iran) . StartinAceramic sites in western Iran have been poorly documented so far, and our knowledge of prehistoric populations in this area derives mainly from surveys and excavations undertaken in the 1950s and 1960s –41. TherExcavations at Chogha Golan revealed 8 m of deposits consisting of 11 archaeological layers (AH XI–I) and one geological horizon at the lowest level of the stratigraphic sequence spanning in all more than 2.000 years. The profile of the deep sounding has been consistently dated, with 17 AMS dates ranging from roughly 11.800 BP  to ca. 9.600 BP (AH I) . With thThe extraordinary richness of the archaeological sediments in artefacts and bioarchaeological remains at Chogha Golan permits a high-resolution investigation of the development of the cultural sequence, subsistence behaviors and the local environment. Large quantities of lithic debitage demonstrate the intensive nature of the activities of the prehistoric community.The development of the lithic industry over the sequence of 11 archaeological layers is relatively indistinct and may indicate relative stability in tool usage. All layers can be characterized by the systematic production of large numbers of bladelets and tools made on bladelets , 56. TheThe site's inhabitants routinely exploited locally available raw materials as well as a very low percentage of imported obsidian. In archaeological horizon II and subsequently AH I obsidian occurs for the first time and comprises approximately 0.5% of the lithic material . The pre in situ preserved ground stone implements are from AH X, most of the mortars, grinding slabs and pestles were recovered from AH III onwards, and were likely used for processing foods. The available data, however, show an emphasis on pounding and little emphasis on grinding. This tendency could be related to the low volume of sediments excavated thus far, rather than an emphasis on pounding activities during the upper occupation. The presence of asphalt and pigments on ground stone tools also demonstrate that they were not exclusively used for food-processing activities. In fact, the available records fit the observation that ground stone tools were often multifunctional devises during the aceramic Neolithic.The first appearance of mudbrick walls, plaster floors and associated ground stone implements such as mortars, pestles and grinding slabs is recorded for AH X. Mortars and grinding slabs were at times found embedded in structure floors showing that they were permanent fixtures in the architecture of the site, while other mortars and grinding slabs were mobile , 62. AltA total of 62 clay small objects from stratified deposits were recovered during the course of the 2009 and 2010 field seasons at Chogha Golan. Most clay objects are from ash and midden deposits. They are found with debris from daily activities and are not limited to obvious ritual contexts. Chogha Golan has provided evidence for the use of clay from the 10th millennium cal. BC onward. The clay usage in Chogha Golan is first evidenced by small and simply formed objects. The usage of clay increases through the sequence, probably reflecting social, economic and cultural development. Excavators recovered 10 clay animal figurines. While an unknown animal head and a goat horn were found from AH VI and III respectively, most of the animal figurines representing sheep/goat, cattle and pig were recovered from AH II and I.Preliminary results of the ongoing archaeobotanical analyses suggest that cultivation of wild plants in the Zagros started equally early as at other places in the Fertile Crescent , 63, butThe Modern and Early Holocene Environment of the Region. The present-day climate of Iran is marked by extreme continental conditions ; the observed mesoclimatic heterogeneity is attributed to the region's complex topography. The strong north-south contrast in atmospheric pressure is due to Iran's position between summer dominating north-east trade winds and the winter dominating westerlies )For the data analysis we categorize individual taxa into groups based on body size and predator evasion tactics (following ), becausδ13C values in cereals provide a drought stress signal when the amount of water received during the grain-filling period is low [δ13C into account, a target of minimum measurements of six individual grains per archaeobotanical sample was taken. Well-developed grains were chosen to guarantee the exclusion of δ13C values that would not reflect the average growing conditions, such as values obtained from immature grains. To eliminate inputs from sedimentary carbonate, the barley grains were reacted with 0.5 M HCl before measurement. The measurements were carried out at the Institute of Geosciences of the University of Tubingen, Germany on a FinniganMAT252 gas source mass spectrometer with a ThermoFinnigan GasBench II/CTC Combi-Pal autosampler.Stable carbon isotope ratios were measured on 159 wild-type barley grains from the site. Measuring stable carbon isotopes in archaeobotanical remains is an established method for identifying past environmental conditions for plant growth in arid and semi-arid environments, as d is low –100. Takδ13C VPDB (Vienna Peedee belemnite ‰) was applied to the measurements of 13C/12C ratios to calculate δ13C in the barley grains. Changes in atmospheric CO2 concentration (δ13C air) over time were considered by calibrating the δ13C from ancient barley into Δ13C values using the approximation AIRCO2_LOESS [The common standard of O2_LOESS .While 110 seed taxa are recorded for the entire assemblage, only up to 38% of these occur simultaneously in one of the archaeological horizons. Some of the taxa are represented in high numbers, such as the small-seeded Poaceae and Fabaceae, goatgrass and barley and thus characterize the different archaeological layers. Only 16 taxa occur throughout all the 11 archaeological layers, almost exclusively consisting of grasses and pulses , the genome donor to free-threshing wheat species, and wild barley (Hordeum spontaneum) are very numerous from the beginning of the occupation. While wild barley decreases in relative proportions throughout the entire sequence, goatgrass appears abruptly reduced in layers V and IV. These trends are partially reflected in the potential arable weed species. These however increase from layer III onwards together with the wheat species (Triticum spp.). Large-seeded pulses, such as lentil (Lens sp.) and vetch (Vicia sp.) occur in lower proportions with a peak in layer VII and then continuously decrease until layer II. Small-seeded grasses strongly increase in layers V and IV and out-number all other taxa.The main taxa groups show distinct developments over the stratigraphic sequence . Large-sWhen the dominant small-seeded grass taxa are excluded from the assemblage, the proportion patterns change . The mosPresuming that the percentages of taxa groups in the different layers are representative for the subsistence development of the entire site, shifts in plant use may be evident. Notable changes are visible in layers V and IV with decreasing proportions of large-seeded grasses, mostly represented by goatgrass, and an increase in proportions of barley and small-seeded grasses.Size development of barley grains has been documented throughout the archaeological horizons . Seed siTriticum dicoccum), corresponding with an increase of potential arable weed taxa and a decrease in goatgrass.At the current stage of the analysis the earliest presence of unequivocally domesticated-type taxa occurs from layer II onwards with the presence of phenotypically domesticated emmer wheat chaff (Ovis or Capra sp.), gazelle (Gazella gazella), pig (Sus scrofa), red deer (Cervus elaphus), and cattle (Bos sp.) are most common. Other taxa include tortoise (Testudo sp.), hedgehog (Erinaceus europaeus), red fox (Vulpes vulpes), Eurasian lynx (Lynx lynx), and unidentified medium-sized birds.Ungulates are the most commonly identified group, followed by fish , which aLarge game, in particular medium ungulates such as sheep and goat, dominate the assemblage by mass throughout the sequence. There is a spike in small ungulates  in horizons V and IV, which correlates to the changes of the archaeobotanical assemblage. Bone weight values of extra-large ungulates  increase in horizon II, corresponding with the appearance of domesticated-type emmer chaff.There are no temporal trends in the proportion of small to large game by mass through the sequence, nor are there changes in the small game component itself. There is, however, a statistically significant trend in the increase of cattle in later phases of the sequence . Pistacia-Amygdalus woodland and riparian vegetation habitats dominated by Salicaceae and Tamarix , both of which might have been associated with riparian woodland habitats. The presence of Hippophaë is limited in the earliest sampled horizons . This taxon is considered by Zohary as tolerant of arid and cold conditions and its presence might represent a residual element from vegetation communities that were locally widespread during the Younger Dryas [ Vitex (a riparian shrub), Prunus , Maloideae , Acer (maple) and various shrubs . Fragments of grass stems  were also very occasionally present in the examined samples . With the exception of Vitex, Ephedra and Lamiaceae the remaining wood charcoal taxa are likely to have derived from vegetation catchments located at some distance from the site. In any case, their rarity suggests that they were not routinely and/or intensively collected as fuel wood.The analysis of the anthracological remains points to the existence of two major arboreal vegetation catchments in the vicinity of Chogha Golan: the semi-arid Tamarix . Chenopoer Dryas . Other v Pistacia-Amygdalus woodland was present and routinely used as a source of wood at Chogha Golan throughout the roughly 2000 years long habitation of the site, from its earliest sampled levels dated to the last phases of the Younger Dryas to the 10th millennium cal BP. Pistacia and Amygdalus charcoals account for >35% of charcoal sample composition in AH XI, AH IX-VIII . A decrease in both taxa is observed from AH VII and is more pronounced in AH VI. Pistacia values pick up very quickly in AH V (rising from 6.8% in AH VI to 21.67% in AH V) and continue to increase through the remainder of the sampled sequence, reaching 90% by AH I . Overall, the wood charcoal samples corresponding to the much shorter time period represented by AH VII–IV  are dominated by riparian taxa . The wood charcoal samples derived from the chronologically latest part of the sequence  indicate yet another shift in sample composition from AH III, still dominated by Salicaceae, to AH II-I that are overwhelmingly dominated by Pistacia.The variations observed in the ubiquity (sample presence) and frequencies  and drought stress (below 16‰) . The mea13C data do not indicate extreme drought stress signals in any of the sampled horizons, they suggest fluctuations of the moisture availability with reduced moisture levels available for plant growth particularly in horizon VI, which may have resulted in the reduction of or regional shifts in some plant populations.Although the Δ2 concentration.Various reasons have been discussed as influential factors on the development of domesticated species, attributing more influence to either humans in relation to their degree of consciousness and socio-cultural needs or to fluctuating natural conditions, such as moisture and temperature increase after the Younger Dryas or increasing atmospheric CO Artemisia pollen as reflected in the Zeribar records that are synchronous with AH XI throughout AH IX at Chogha Golan, suggest increases in moisture. This is in agreement with the size development of barley grains and the δ13C record from barley  vegetation disturbance by hunters and/or herders has been suggested for the Lake Zeribar region. For this horizon the lowest Pistacia charcoal proportions have been documented, while Salicaceae and Tamarix percentages are increased. Drought and/or lowered biomass productivity may have limited the efficiency of plant management, which might also have resulted in a temporary change in animal prey availability. The latter becomes apparent in AH V with increased bone weight for gazelle. This is accompanied by large numbers of small-seeded grasses, a strong reduction in goatgrass and a relative peak in wild barley that may have been intensively cultivated under sufficient moisture availability in relation to the previous shortcomings in AH VI. Evidence for the intensified cultivation of wild barley is supported by increased grain size mean values. The conditions are unchanged in AH IV when first domesticated-type rachis segments of barley appear alongside a possible clay animal head.Before the earliest occupation of the site, temperatures increased with the end of the Younger Dryas. High evaporation and low precipitation led to low lake levels, water salinization and scattered occurrence of a very thin arboreal cover, dominated by Chenopodiaceae. During the time of the first settlement horizon (AH XI), conditions were still relatively cold and dry, as indicated by the occurrence of Pistacia pollen increases in a relative sense and oak pollen is also present at Lake Zeribar. This correlates with wood charcoal finds from Chogha Golan which show a steep increase in Pistacia percentages. In this horizon the first domesticated-type emmer chaff and an increase in cattle bone is documented. The first animal figurines representing cattle also occur in this horizon, as well as obsidian that comprises 0.5% of the lithic material. In AH I Pistacia charcoal increases further, and mortars, pestles and grinding slabs are frequent. Overall, the environment-subsistence-coupled developments suggest that despite the moderate fluctuations in biomass productivity for various reasons, the inhabitants of Chogha Golan created resilient living conditions through an increase in the use of large-bodied ungulates and cereal cultivation.In AH III the first evidence for the use of dung fuel is represented in the remains of a floor and suggests the intentional collection of dung for burning or construction, or increased access to dung from keeping certain species at the site. In this layer, grain size mean values and proportional percentages of wild barley decrease, whereas in the following layer AH II an increase of arable weed taxa and wheat species is apparent. Pistacia  register high values (percentage frequencies and ubiquity) in AH I-II. Pistacia and Amygdalus also have high values in the earlier part of the sampled sequence (AH XI–VIII). It is therefore possible that a different spatial pattern of fuel use and deposition of fuel debris is reflected in charcoal sample composition from AH VII–III .Although the main seed and faunal taxa groups from Chogha Golan are relatively stable in composition throughout the archaeological layers, changing proportions of taxa in the middle (AH V and IV) and the late (AH II) parts of the sequence are considerable. While most of the archaeological horizons are dominated by large-seeded grasses, that is, goatgrass and barley, layers V and IV contain large amounts of small-seeded grasses and show signs of intensification of barley cultivation . The firPreliminary micromorphological analyses have indicated that middens, as revealed throughout AH VII–III were likely accumulated inside abandoned structures . Althoug13C values in barley.A major concern in interpreting our data is the question of the representativeness of a 1 m² excavation area, and whether the fluctuations described above, particularly in AH V and IV, indeed indicate a shift in resource use or subsistence, or if the patterns only reflect the limitations of the excavation. Synchronous shifts in the proportions of some important plant and animal taxa, the correlation of barley proportions with grain size development, as well as the appearance of domesticated-type barley rachis internodes in AH IV and domesticated-type emmer chaff in AH II support the patterns as representative. The wood charcoal data, however, do not correlate with the changes in the seed and faunal assemblages. Instead they are in relatively good agreement with the ΔFind densities of the different find groups may partially clarify the complex relationship of taphonomy and contextual differences in the different materials recovered from the deep sounding .The general trend of the find densities is very similar in all of the groups and may indicate underlying contextual aspects of the deposits. Except for animal bones, find densities of the other categories are high in AH XI and decrease until AH IX, though densities of faunal remains must be examined with caution due to the differential wet screening methods mentioned above. Charcoal, animal bones and lithic artefacts show their peak in find density in AH VIII. The densities of all find categories decrease after AH VIII, stagnate or slightly increase in AH VI and V, and again peak in finds in AH IV. After AH IV there is a continuous decrease in find densities in all categories, except for chaff remains, indicating their intense production in the settlement. In AH I the find densities are particularly low as a result of taphonomic processes in close proximity to the soil surface.As concerns the specific question of AH V and IV reflecting some kind of bottleneck in subsistence, the high find densities in all categories suggest that a bias through selective preservation of the more fragile organic remains can be excluded .The long-term subsistence reliance on cultivated wild plant species and wild game, with the first perpetual domesticated-type emmer appearing after almost 2.000 years of occupation suggests, despite a short-term depression in horizons V and IV, a generally proliferous environment, rich in resources at Chogha Golan.13C signals of increasing moisture availability. The slight drought stress signal in AH VI, which can be easily understood as a plausible effect of inter-annual variation of precipitation, is followed by changes in the plant and faunal assemblages in layers V and IV. These layers show increased proportions of small-seeded grasses and gazelle bone, and a relative increase of wild barley proportions and its grain size. In AH IV, roughly around 10.200 cal BP barley rachis internodes also show domesticated-type features that disappear again in the following horizons. The short-term appearance of domesticated-type characteristics in barley suggest that the inhabitants at Chogha Golan were about to domesticate barley around 10.200 cal BP, but continued with the earlier practice of plant use (i.e. probably introducing again more wild types). Grain size and Δ13C signals decrease again in AH III. With the transition to AH II further changes become apparent that include an increase in Triticum species, including phenotypically domesticated emmer wheat chaff (Triticum dicoccum), increasing percentages of potential arable weeds and increasing numbers of cattle bones.The development of barley through time supports the model of protracted domestication. Both proportions and grain sizes of wild barley increase from AH XI to IX and correlate with ΔIn our previous work about Chogha Golan we argued that domestication of emmer may have evolved on-site , supportHypotheses concerning the transport of grain from elsewhere, such as the western Fertile Crescent, can indeed be used to explain a protracted development of domesticated species. However, this argument is less convincing in a proliferous environment offering sufficient resources, and thus enabling a resilient way of living. Given the fact that there is evidence for the extensive use of plants, we may question whether the development of agriculture really required significant changes in human subsistence behavior or whether these changes were gradual and appear to be substantial only from our modern perspective.Overall, the entire stratigraphic sequence at Chogha Golan can be seen as a continuously increasing intensification in the use of grasses, even in layers V and IV. However, a bottleneck in the availability of large-seeded grasses might have occurred during the formation of these two strata, which was attenuated by the increased gathering of small-seeded grasses, shift to small-bodied ungulates and intensified cultivation of barley, followed by emmer cultivation in the subsequent phases.


Plasmodium impose on human health worldwide the enormous burden of malaria. The possibility to genetically modify several species of malaria parasites represented a major advance in the possibility to elucidate their biology and is now turning laboratory lines of transgenic Plasmodium into precious weapons to fight malaria. Amongst the various genetically modified plasmodia, transgenic parasite lines expressing bioluminescent reporters have been essential to unveil mechanisms of parasite gene expression and to develop in vivo imaging approaches in mouse malaria models. Mainly the human malaria parasite Plasmodium falciparum and the rodent parasite P. berghei have been engineered to express bioluminescent reporters in almost all the developmental stages of the parasite along its complex life cycle between the insect and the vertebrate hosts. Plasmodium lines expressing conventional and improved luciferase reporters are now gaining a central role to develop cell based assays in the much needed search of new antimalarial drugs and to open innovative approaches for both fundamental and applied research in malaria.The unicellular protozoan parasites of the genus  Plasmodium infect humans, with Plasmodium falciparum and P. vivax causing over 200 million cases/year and P. falciparum inflicting virtually all the 6–700,000 annual deaths (2013) recorded mainly in children of Sub-Saharan Africa.Half of the world population is at risk of malaria , the mosAnopheles mosquito and a vertebrate host . When an infected female mosquito bites a human, the Plasmodium sporozoites travel to the liver and invade hepatocytes, where parasites replicate as hepatic schizonts until several thousand merozoites are produced and released in the bloodstream. In P. vivax, but not in P. falciparum, some liver parasites remain instead quiescent (hypnozoites), resuming replication, and infection after several weeks or months. Upon erythrocyte invasion in the bloodstream Plasmodium parasites undergo asexual replication forming mature schizonts whose rupture releases merozoites that invade new erythrocytes. Some blood stage parasites differentiate instead into male and female gametocytes that, when ingested in the mosquito blood meal, are activated to produce gametes. Gamete fusion in the insect midgut produces a zygote which develops into a motile ookinete, traversing the gut wall, and transforming into an oocyst, where 1000s of sporozoites are produced. The life cycle is closed when sporozoites, migrated from the ruptured oocyst to the mosquito salivary glands, are injected in a new human host by the insect bite.The malaria parasite exhibits a complex life cycle involving an P. falciparum malaria, the ability of parasites to sequester in the microvasculature of several organs, including the brain, is a major cause of disease severity, and of a fatal outcome  luciferase gene whose expression, driven by the promoter of a parasite sexual stage-specific gene, was measured in ookinetes of the bird parasite P. gallinaceum  enabled simultaneous detection of the two parasite produced reporters .Luciferase enzymes catalyze the light-producing chemical reactions of bioluminescent organisms, in which a luminogenic substrate  is oxidized in the presence of ATP, yielding photons. These can be accurately measured by a luminometer with a sensitivity and a virtual absence of background that made bioluminescent reporters potent and versatile tools in biology . Luciferlinaceum . Subsequarasites , includieporters . Since tasmodium , luciferPlasmodium, and elucidating the biology of this process may therefore lead to design novel malaria transmission-blocking strategies. Some studies with bioluminescent parasites highlighted the importance of post-transcriptional regulation acting on stability and translation of several mRNAs, including those encoding major proteins of the gamete and ookinete surface  in mosquito stages  the mean luminescence intensity of individual and pooled mosquitoes accurately quantified mean oocyst intensity, eliminating the need for mosquito dissection, and putting the basis for significant SMFA scalability  and in vivo imaging system (IVIS) in the analysis of parasite liver stage development in live mice and in cultured hepatocytes. Real-time BLI requires injection of the luciferin substrate in the mouse or in the dissected organ and an intensified charge-coupled photon counting video camera to measure photon emission  gene  gene  or the iar genes , or evalere able , or unabere able  to recru evasion . In summ evasion , but als systems  and to t systems .P. falciparum, has been the screening for new antimalarial drugs, an endeavor that the appearance of artemisinin resistance in South East Asia makes dramatically urgent. In the past decades, in vitro methods measuring the incorporation of [3H]-labeled hypoxanthine and ethanolamine or the activity of parasite Lactate Dehydrogenase have been the standard for P. falciparum cell based assays and used in large drug screenings  gene promoter in asexual stages enabled establishment of a cell-based luciferase drug screening assay in 96w plates (5–106 parasites per well (P. berghei parasites expressing a firefly luciferase-GFP fusion were used for in vitro and in vivo bioluminescence drug assay, enabling use of animal models to test new drugs in vivo (A major effort in the fight against malaria, particularly reenings . The demarasites . To thisw plates , subsequper well . Also P. in vivo .Plasmodium gametocytes are the parasite sexual stages responsible for the transmission from the vertebrate host to the mosquito. Male and female gametocytes are formed in the bloodstream and, in P. falciparum, they mature in 10 days through five developmental stages. Upon ingestion in the mosquito gut, mature gametocytes promptly differentiate into gametes and fertilization ensures parasite infection in the insect vector. A key priority in the present goal to globally eliminate malaria is to identify new drugs targeting in the bloodstream both the asexual and the sexual stages of the parasite. However, the non-replicative nature of gametocytes imposed to develop specific cell based screening assays, different from those used for asexual stages. One problem is for instance that of false negative signals due to the persistence of fluorescent reporter or of parasite enzyme activities in unhealthy or dying gametocytes. P. falciparum lines expressing a GFP-firefly luciferase gene under gametocyte specific promoters were established (P. falciparum of the use of a potent luciferase from Pyrophorus plagiophthalamus under a gametocyte promoter enabled to perform for the first time BLI at the level of single parasite cells, individually distinguishing live and dead P. falciparum gametocytes (ablished  and usedablished . Recentlablished . In thisetocytes .P. falciparum beyond the P. pyralis and Renilla enzymes (Virtually all stages of the complex life cycle of malaria parasites have been enlightened by the use in several studies of luciferase reporters. These engineered parasites provided key answers to fundamental biological questions and now represent important tools for drug screening. Novel potent reporters have already expanded the luciferase repertoire used in  enzymes , increasP. plagiophthalamus were expressed in P. falciparum immature and mature gametocytes, providing for the first time the possibility to simultaneously measure differential, stage specific effects of drugs in a dual-color luciferase assay, and opening the possibility to apply multicolor bioluminescence to any parasite stage in fundamental and applied studies. A dual expression system with distinct luciferases would for instance be valuable in cell based high-throughput screenings to readily identify and discard compounds active against the reporter rather than the target cell (The possibility to tune luciferase emission properties, such as emission wavelength, kinetics or termo- and pH-stability, via random or site-directed mutagenesis or use of enzyme natural variants, led to introduce multicolor bioluminescence in antimalarial drug screening. A green and a red light emitting luciferase from get cell , as theyP. pyralis enzyme (P. falciparum lines which express a brighter deep-sea shrimp luciferase equipped with sequences driving the reporter in the parasite cytoplasm or in erythrocyte compartments (Another promising application of luciferase reporters is through their fusion to specific signals used by the parasite to traffic proteins in different extra cellular compartments of the infected erythrocyte. As protein export is uniquely regulated in the parasite and is essential for its survival, use of such fusions may be invaluable to screen for compounds targeting this process. Preliminary studies were conducted with the s enzyme  and moreartments .P. falciparum stages and human cell types in vitro can provide new insights of the physiology of asexual and sexual stage parasite sequestration. The increased sensitivity achieved in in vivo mouse imaging with a red-shifted luciferase expressed by the unicellular protozoan parasite Trypanosoma brucei (Plasmodium infected mice. Importantly, the increasing availability of humanized mouse models for P. falciparum and P. vivax infections, supporting development of asexual, and sexual blood stages and of liver stages (P. falciparum transgenic lines with a luciferase expressed constitutively (In another field of application, the achievement of single parasite cell BLI and the availability of luciferases whose red-shifted light emission is more efficiently detectable from blood and tissues are paving the road to significant progress in analyses of the host-parasite interplay. Co-cultures of different a brucei  is promir stages  and the tutively  or underThe wealth of biological information provided by the use of engineered bioluminescent malaria parasites, not to mention those not reviewed here expressing a variety of fluorescent reporters, has been and will most likely continue to be enormous. The confined use of these whole cell biosensors in laboratory settings does not pose regulatory concerns on environmental release. From their aseptic sites of utilization, these genetically modified parasites will nevertheless have the most significant impact in the real world, contrasting the unbearable burden of a worldwide devastating disease.GS drafted and PA edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.