In basic research, the etiology of fear-related pathologies, such as post-traumatic stress disorder (PTSD), is conceptualized using fear-conditioning protocols that pair environmental stimuli  with an aversive, unconditioned stimulus (US) to elicit an assessable conditioned fear response. Although pathophysiological models agree that regulatory dysfunctions in this associative process may instigate fear-related pathology, current opinions differ in regard to the nature of these dysfunctions. Primarily derived from studies in rodents, the prevailing perspective proposes that pathological fear-reactions develop from intensified and overly consolidated CS-US associations. Alternatively, models derived from studies in humans suggest that tempospatial inaccuracies in representations of associative fear might precipitate pathology by engendering failure to differentiate present experiences and past memories of threat. To test this concept in rodents, we administered rats with cognition enhancing doses of Methylphenidate before or after fear conditioning and measured long-term alterations in their conditioned fear behaviors and PTSD-like reactions. The administration of Methylphenidate before fear-memory formation indeed reduced anxious-like responses during fear-memory retrieval one month later. An individual profiling analysis revealed that Methylphenidate onset had opposing effects on the risk for PTSD-like classification. The modulation of initial learning and formation of associative fear normalized the risk for developing PTSD-like reaction. In contrast, when the effects of Methylphenidate were exerted only over later consolidation this risk increased markedly. When examined under current psychiatric and neuropharmacologic literature, these results reveal a possible strategy of using low-dose Methylphenidate for the prevention of PTSD in high risk populations. Pathophysiological models identify the amygdala, hippocampus and medial prefrontal cortex (mPFC) as key constituents in the neural networks mediating this classical learning, and generally agree that regulatory dysfunctions in associative processing within this circuitry are at the core of pathological fear behavior occurring in fear-related disorders.8 However, current opinions differ in regard to the nature of the dysfunction and its specific effects on fear memory formation and modulation.Fear learning is an adaptive process that allows stimuli preceding an aversive outcome to acquire the capacity to signal danger and elicit defensive responses against potential future threats. Although essential for generating appropriate responses in altering environments, this memory process is highly sensitive to increasing levels of stress.7 producing overly consolidated and potentially pathogenic representations of associative fear.4 With time, this associative imbalance is suggested to impair CR regulation and mPFC capacity to inhibit excessive fear-responses to the CS features.7Centering on the amygdala as a site of CS–US convergence and initiation of fear response, the dominant view asserts that enhanced acquisition and consolidation of aversive associations precipitate the development of fear-related disorders. Primarily derived from fear conditioning studies in rodents, this viewpoint proposes that exaggerated amygdala responses to aversive inputs critically strengthen CS-US pairings,8 and in fear-related disorders, induce critical changes in core representations of the CS space.9 Such studies give rise to an alternative perspective that emphasizes the role of the memory’s accurateness in the pathogenesis of fear-related disorders.10 According to this perspective, aversive US inputs modify basic processes of associative encoding during emotional learning, and in extreme situations, impair the precision of tempospatial representations via hippocampal plasticity.11 Considering the role of the hippocampus in pattern separation,12 these imprecisions are suggested to engender failure to differentiate present experiences and past memories of threat and thus provoke pathology in liable individuals.11 Yet, this alternative perspective lacks the resolution and causativeness provided by animal research. To bridge this gap, we have recently developed a reverse-translation model that enables to identify dysregulated patterns of fear-behaviors and PTSD-like reactions in rodents.13 In this model, rats are initially habituated to the context of the water associated zero maze (WAZM) for 4 consecutive days and form a safe representation of its environment. Following the 4th day habituation, rats are exposed to an extremely aversive underwater trauma (US) in the aquatic center of the WAZM and form a new fear-associated representation for its context (CS). One month later, rats are presented with this CS to measure long-term alterations in different fear responses and an individual profiling approach is then implemented to classify PTSD-like individuals. Combining this novel protocol with microanatomical analyses of cellular activation, we uncovered an extremely anxious sub-group, within the stress-exposed population, demonstrating dysregulated patterns of excitation and co-activity in the mPFC–amygdala–hippocampus circuit.13 Importantly, these patterns exposed a distinct imbalance within the hippocampus of the anxious individuals, indicating an intrinsic desynchronization during fear memory retrieval between its dorsal division (DH), which subserves tempospatial memory processing,14 and its ventral division (VH) which mediates fear memory and anxiety.14Deviating from these conventional conceptions, recent findings of neuroimaging studies in humans provide evidence that aversive inputs can modulate corticolimbic processing of the CS features during conditioning,13 seemed to concur with the alternative perspective above, we set out to evaluate its predictions by combining our behavioral model with pharmacological manipulations of fear memory formation. To this end, we took advantage of Methylphenidate (MPH), a selective dopamine (DA) and noradrenaline (NA) reuptake inhibitor that is commonly prescribed for the treatment of attention deficit hyperactivity disorder (ADHD). Although MPH increases the expression of extracellular catecholamines, at clinically relevant low doses it can improve cortical cognitive function while exerting only modest effects in neural circuits associated with arousal and pleasure.15 In nonclinical samples, low-dose MPH was shown to improve working memory performance and consolidation of long-term representations in both healthy individuals18 and laboratory rats.20 These low-dose cognition-enhancing effects are suggested to result from strengthening of signal processing within the PFC,15 and synaptic plasticity within the hippocampus,21 via preferential activation of different NA (α/β) and DA (D1/D5) receptors.As our neuroanatomical data, correlating fear-related pathology with tempo-emotional impairments in hippocampal representations of aversive associations,19 we administered MPH 1 h before, or after, the safe context of the WAZM was paired with the underwater trauma experience. Given its memory enhancing effects, we hypothesized that MPH administration before the acquisition of this aversive association may reduce inaccuracies in its tempospatial–emotional representation, and by this, reduce pathological responses to its reminders in the long-term.To examine whether the accuracy of representations of an aversive association contributes to the development of fear-related pathology, in this study we treated rats with procognitive doses of MPH (0.5 mg/kg P.O.) at different stages of fear-memory processing. As in rats extracellular catecholamines reach peak concentration within 40 min of oral MPH onset,n=16 each). As illustrated in n=20 in each condition): no treatment (NT), Vehicle, Pre-MPH and Post-MPH. Rats were then habituated to the WAZM test and the oral administration of MPH and vehicle for 4 consecutive days. In the Pre-MPH condition, MPH was administered 1 h prior to the WAZM tests. In the Post-MPH condition MPH was administered 1 h after the WAZM tests. In the Vehicle condition, vehicle solution was administered 1 h prior to the WAZM tests. Habituation procedures were performed between 1000 and 1700 hours. Immediately after the 4th day test, 50% of the rats in each condition were randomly assigned to the underwater trauma (UWT). In this trauma condition rats (n=40) were exposed to the WAZM test and immediately after to the UWT stress of 45 s restrain underwater in the WAZM center. In the control condition rats (n=40) were only exposed to WAZM test. The UWT exposures were consistently conducted between 1100 and 1400 hours throughout the experiments. One month later all rats were re-exposed to the WAZM testing as a contextual reminder by a blind investigator. On the basis of their behavior rats were classified as ‘PTSD-like’ individuals as described below.All experimental procedures were performed adhered to the NIH Guide for the care and use of laboratory animals and were approved by the University of Haifa ethical committee. This study used 80 adult male Sprague Dawley (SD) rats (8–9 weeks old) weighing 250–275 g  at arrival. Animals were housed two per cage in a temperature-controlled (23±1 °C) animal facility on a 12:12 h light–dark cycle (lights on 0700–1900 hours). They had ad libitum access to standard rodent chow pellets and water. Throughout the study, identical procedures were repetitively performed for five times on consecutive batches .MPH and vehicle were orally administered during the initial 4 days of the experiment. For the MPH treatment (0.5 mg/kg), 10 mg Ritalin tablet  were grounded and fluidized in 10% sucrose in tap water solution to a 1 mg/ml concentration. For the vehicle, only the 10% sucrose in tap water solution was used. The solution (~75 μl) was then administered with a micropipette by placing the filled tip in or near the animal’s mouth and slowly discharging the suspension to allow the animal to drink . This apparatus enables the formation of an association between the maze and an underwater trauma, and by that, the assessment of complex behaviors during re-exposure to the context that immediately precedes a traumatic experience. For the tests, rats were first habituated to the room for 2 min and then were placed into one of the open arms facing a closed part of the apparatus. Rats were allowed to explore the arena for a 5 min session. During this time rat’s behavior was tracked, recorded and analyzed by the Etho-Vision system . Behavioral measures included the time spent in the open arms, distance traveled in the open arms, distance traveled in the closed arms and total freezing. Standard anxiety indices  and submerged under water for 45 s, using a special metal net (20 × 10 × 15 cm). After the procedure the animals were dried briefly and returned to their housing cages. This procedure is known to acutely and enduringly increase anxiety-like behavior13 For this, anxious-like responses were categorized according to the distribution of the behvior of the no-treatment, no-trauma, group (Control NT) during the WAZM re-exposure. Classification criteria were based on the lower 20th percentiles of Control NT's distribution for time spent in the open arms, distance traveled in the open arms and distance traveled in the closed arms. The freezing criterion based on the upper 80th percentile of control NT's distribution. All rats were individually discerned for each criterion. For the final classification, every rat that demonstrated a behavioral profile that fell within a minimum of 3 out of 4 criteria was classified as a PTSD-like rat.This individual profiling analysis based on the within group variablility of animals behavior in order to identify behavioral reactions of dysregulated fear as was shown before.post hoc comparisons.Data were analyzed using the IBM SPSS Statistics Software . Shapiro–Wilk test of normality and Levene’s test for homogeneity of variance were utilized for inclusion in parametric tests. No animals were excluded from the behavioral analyses. A 2 × 4 × 3 multivariate analysis of variance (MANOVA) was used to examine the effects of UWT (Trauma/Control) and treatment  on the standard anxiety indices of time, distance and percentage of freezing. Significant interactions were followed by Bonferroni λ=0.543, F=19.62, P<0.001), the pharmacological treatment =2.37, P=0.015) and their interaction =1.98, P=0.045). As shown in P<0.05, Bonferroni post hoc), the Pre-MPH trauma-exposed rats exhibited a similar to the control groups choice of risk behaviors as measured by the anxiety indices for the ratio of time spend in the safe arms =28.9, P<0.001), with 60% of trauma-exposed rats in the no-treatment and Vehicle conditions qualifying for PTSD-like classification. The pharmacological treatment however induced a differential effect . Classification criteria based on the lower 20th percentiles of Control NT's distribution for time spent in the open arms (<59.4 s), distance traveled in the open arms (<295 cm) and distance traveled in the closed arms (<507 cm). The freezing criterion based on the upper 80th percentile of Control NT's distribution (>30.8 s). All rats were individually discerned for each criterion and every rat that demonstrated a behavioral profile that fell within a minimum of 3 out of 4 criterions was classified as a PTSD-like rat . In linel effect . The adm15 of MPH before an aversive experience may acuminate its associative representations and consequently reduce pathological responses to its reminders. Supporting this hypothesis, our data demonstrated that administration of MPH before the encoding and consolidation of a traumatic experience actually reduces pathological fear responses to its associative reminders in the long term. When combined with the abnormal hippocampal patterns we have previously identified in highly anxious rats,13 our results emboldens the theoretical perspective proposing that incoherent representations of sensory modalities and emotional valence can promote less regulated reactions to trauma related cues30 and, as a result, fear-related pathology.11 Moreover, in line with this pathophysiological concept, our individual profiling analysis revealed that the Post-MPH had an opposite effect on the risk for PTSD-like classification. Thus, when the initial formation of associative fear representations was modulated by Pre-MPH, the risk for developing PTSD-like reaction was normalized. Yet strikingly, when the initial learning was not manipulated, and the effects of MPH were exerted only over the consolidation of the aversive representations, the risk for developing PTSD-like reaction increased markedly.To test if inaccuracies in the representations of aversive associations might contribute to the development of fear-related pathology, we combined a reverse-translation model of PTSD in rats with pharmacological manipulation of fear memory formation. We hypothesized that administration of cognition-enhancing doses7 yet here we used the WAZM, an apparatus that enables multiple behavioral measures during fear memory retrieval.23 Thus, had we solely relied on freezing assessment no substantial effects for Pre-MPH could be concluded, as all trauma-exposed rats demonstrated a significant increase in freezing in comparison with controls. Moreover, in the absence of this main effect on freezing, the results of the anxiety indices would be limited by the possible risk for Pre-MPH state dependent effects. Yet, the WAZM enabled us to assess reflexive freezing to ascertain that fear learning indeed occur, and as an indication of increased arousal, and in parallel, to assess additional behaviors that may be more relevant for indicating re-experiencing and avoidance-like symptoms.23 Finally, although providing priceless mechanistic insights into the circuitry underlying fear learning and memory, the majority of studies in rodents contrast exposed and non-exposed populations while individual variations within the exposed population draw less attention.13 Translationally wise, this is at odds with clinical data showing that only a subset of trauma-exposed individuals develops PTSD in the long term.13 Our results thus emphasize the importance of accounting for inter-individual differences to better understand the psychophysiological processes transforming a ‘normal’ fear behavior to a pathologically dysregulated fear response.When considered under the common views in the field of behavioral neuroscience, our results reveal a possible inconsistency. We found that facilitation of memory formation during a fear conditioning protocol did not increase the expression of conditioned fear responses in the long term . Nonethe31 However, to the best of our knowledge, this study provides first evidence that pre-trauma low-dose MPH may possibly prevent post-traumatic fear-related pathology. Furthermore, to avoid any reinforcing or anxiolytic effects, as seen with higher doses of MPH, here we used very low doses  to exert qualitatively different, memory-enhancing effects with strong translational relevance.15 Regrettably, due to its psychopharmacological nature, our data cannot provide direct indications of the neurocircuits that could underlie the pre-trauma MPH amelioration of psychopathological post-traumatic symptoms. Nonetheless, the neurofunctional effects of low-dose MPH strongly associate with the neural correlates of fear-related disorders, particularly the ones involved in PTSD. When combined, independent observations from both humans and rodents can thus offer strong logical interpretations for possible micro- and macro-level mechanisms involved in the preventive effects of pre-trauma MPH.In regard to potential therapeutic mechanisms of MPH in fear-related disorders, it was previously shown that post-exposure chronic administration of high-dose MPH  can improve PTSD-like symptoms in rats long after stress exposure.2 adrenergic and D1 dopaminergic receptors . MPH is also suggested to affect glutamatergic signaling in the PFC via specific alterations in N-methyl-d-aspartate receptor (NMDAR) function.32 In the context of stress, MPH is suggested to restore stress-induced changes in PFC dendritic spine densities and resultant cognitive impairments.33 Thus, although stress exposure disrupts PFC dendritic growth33 and NMDAR-dependent information processing,34 post-exposure MPH was shown to reduce stress-induced impairments in PFC NMDAR32 function and restore cognitive performance.33In rodent research, the effects of MPH have been mostly studied in fronto-striatal circuits. Within the PFC, low doses of MPH are suggested to bias neuronal activation to task-relevant information, while simultaneously reducing responding to irrelevant stimuli, via specific activation of catecholamine signaling at α35 Yet, differing from the PFC pathways, within the hippocampus low-dose MPH was recently shown to strengthen signal transmission ) via specific activation of β-adrenergic and D1/D5 dopaminergic receptors; and promotion of mobilization and insertion of AMPA receptors in synapses involved in recent patterns of activity.21 Given the differential effects of emotional stress on synaptic plasticity in the DH and VH,14 this specific involvement of β-adrenergic receptors in hippocampal LTP is extremely relevant to possible mechanisms involved in the protective effects of pre-trauma MPH. Although under normal conditions the ability to evoke LTP is lower in the VH compared with the DH, acute stress exposure reverse this completely, causing facilitation of LTP in the VH and its suppression in the DH.36 Over-regulating functional VH-amygdala routes during fear memory formation,36 this dynamic plasticity is suggested to consequently produce the abnormal VH-amygdala synchronization involved in dysregulated fear responses during the retrieval of representations of aversive associations.27 Strikingly, the preferential target of MPH in the hippocampus, β-adrenergic receptors,21 are also predominantly involved in this stress-induced routing of synaptic plasticity,38 and specific long-term underwater trauma-induced changes in DNA damage pathways in the VH.39In the hippocampus, stress-induced impairments in glutamatergic signaling have been suggested to contribute to the development of stress-related pathology.41 That is, while upregulation of left-hemisphere dopaminergic function is related to task accuracy and left-PFC control over working memory (WM) processes,42 single low doses of MPH were shown to normalize left-hemisphere dopaminergic underfunctioning in ADHD,41 and to upregulate functional activation in left-PFC regions involved in temporal processing43 and mediation of WM load.40 These lateralized effects become specifically relevant given that functional lateralization is strongly related to emotional regulation45 and memory processing46 in healthy human populations, and the intrusive nature of traumatic recollections48 and severity of symptoms50 in PTSD. Moreover, while in healthy subjects successful regulation of emotional responses during aversive learning involves left-hemisphere activity increment51 and selective left-hippocampus activation modulates the accuracy of its associative representations,8 cued-retrieval of traumatic representations and emotional flashbacks in PTSD were found to involve selective left-hemisphere underactivation48 . A concordant combination of the above can suggest that the lateralized effects of Pre-MPH might balance functional asymmetries involved in the processing of intense fear-emotions and their perceptual co-occurring space during the initial encoding and consolidation of an aversive experience. In consideration of the dual-role suggested for mixed lateralization, both as a risk-factor and the result of irrepressible re-experiencing of trauma-related fear in PTSD , such lateralized-modulations, reducing tempospatial imprecisions in emotional representations, thus might subsequently prevent dysregulated fear-responses to contextual reminders of the aversive event in the long term.At the macro-level, the preventive effects of pre-trauma MPH on fear-related symptoms can be linked to the asymmetric effects of low-dose MPH within the cerebral hemispheres.53 Generally applied immediately after trauma, in most prevention strategies for PTSD attention is typically trained away from threat cues under new safe circumstances.55 However, differing from these common strategies, a recent trial by Wald et al.53 provide evidence that pre-trauma modification of attention to the direction of threat cues can actually reduce the risk for post-traumatic distress in the long-term. Utilizing attention bias modification training as a preventive strategy, this randomized controlled study trained soldiers to attend towards threat, in an attempt to enhance cognitive processing of potentially traumatic events. Indeed, when delivered prior to combat deployment, the pre-trauma attention bias modification training intervention mitigated the risk for PTSD following combat exposure.53 Revealing a pre-trauma window for PTSD prevention, the findings of this study may correspondingly point to the potential mechanisms involved in the preventive effects of pre-trauma MPH found here. In specifics, a possible key involvement of the attentional threat-monitoring system, and its optimal functionality, in the delicate balance between environmental demands and neurocognitive responses during trauma53 and the initial formation of its associative representations. When taken together, the detailed anatomical observations presented above illustrate a complex multidimensional circuitry, which may be involved in the preventive effects of pre-trauma MPH. Nevertheless, in line with the conceptual offer of Wald et al.,53 both micro- and macro-level observations indicate that pre-trauma MPH can selectively modulate basic attentional functions during trauma exposure. This modulation may preattentively bias emotio-cogntive processing towards cues that signal genuine danger. Furthermore, in such a case, the pre-trauma MPH could exert its preventive effects by promoting a more adaptive formation of associative representations of the traumatic experience. Given the temporally restricted impact of MPH and the low dose required for its preventive effects in this study, our findings may suggest a relatively simple low risk strategy for the prevention of PTSD. Particularly in populations such as combat and emergency response personnel that faces high risk for traumatic exposure within a timely defined framework. For example, during combat deployment, low-dose MPH can be selectively administered to soldiers headed for routine engagement in operational assignments.In extreme situations, such as combat, the identification of environmental cues that signal genuine danger is critical. Under such circumstances, directing attention to threat cues may thus become highly adaptive.To conclude, our behavioral data provide evidence that basic memory impairments play an important role in the development of fear-related pathology. This is in line with the hypothesized involvement of tempospatial–emotional inaccuracies in aversive representations in the pathogenesis of fear-related disorders. Bearing in mind the dose-dependent lateralized effects of MPH, and the significant connection between mixed lateralization and PTSD, our results may offer a new pharmacologic strategy of using low-dose Methylphenidate for the prevention of post-traumatic pathology in populations that are at high risk for traumatic exposures.


Paper-based micro analytical devices offer significant advantages compared to the conventional microfluidic chips including cost-effectiveness, ease of fabrication, and ease of use while preserving critical features including strong capillary action and biological compatibility. In this work, we demonstrate an inexpensive, rapid method for high-throughput fabrication of paper-based microfluidics by patterning hydrophobic barriers using a desktop pen plotter integrated with a custom-made, low-cost paper feeder. We tested various types of commercial permanent markers and compared their water-resistant capabilities for creating hydrophobic barriers. Additionally, we studied the performance of markers with different types of paper, plotting speeds, and pattern dimensions. To verify the effectiveness of the presented fabrication method, colorimetric analysis was performed on the results of a glucose assay. Some particularly attractive advantages of paper-based micro analytical devices are affordability, facile disposability, portability, and simple fabrication which facilitate mass-production12. Moreover, multiplexing these devices enables simultaneous execution of multiple assays on a single device without cross-contamination13. Sample transportation in paper-based microfluidics is controlled via patterned hydrophobic barriers and propagated by means of the strong capillary action of paper, obviating the need for pumps14. Several approaches including photolithography15, laser etching17, and plasma treatment18 have been extensively presented with well-characterized materials such as glass, silicon, and polymers for the fabrication of micro analytical devices. The limitations of these fabrication methods, including lengthiness and the need for expensive instruments and trained personnel, pose challenges regarding ease of mass fabrication and cost-effectiveness in both resource-limited regions and developed countries21.Utilizing paper as an easy-to-use and disposable platform for micro analytical devices has garnered considerable attention for point-of-care applications in clinical diagnosticset al. introduced the idea of using paper as a substrate with patterned hydrophobic barriers using a UV-polymerized photoresist15. Abe et al. developed a fabrication method based on inkjet printing for creating hydrophilic channels on hydrophobic paper22. Over the years, researchers have employed different hydrophobic agents including wax24, permanent marker ink11, polydimethylsiloxane (PDMS)25, and alkylketene dimer (AKD)26 for delimiting the barriers. Among these approaches, flexographic14 and inkjet printing27 are promising for high-throughput and mass fabrication of paper-based microfluidics28 due to their advantages of affordability and simple single-step nature12.Martinez 27. Since the intensity of the color in detection zones is a function of analyte concentration, quantification of results can easily be performed with a digital camera13. Therefore, we used digital image processing to locate the detection zones and analyze the color intensities by using a MATLAB image processing script.In this work, we developed a pen-plotter-based approach, integrated with a custom-made, low-cost paper feeder Fig.  for singWe plotted patterns on both delicate task wipers and chromatography paper with all types of markers we used in this study Fig. . MajoritThe results of testing water-resistant capability of markers demonstrate that, only the patterns plotted with Comix broad-tip markers and 3 passes of Deli black markers are able to resist the aqueous solution on the chromatography paper Fig. . HoweverPlotting speed is directly related to the amount of ink that diffuses through the paper. Therefore, we can increase the amount of diffused ink and thereby increase the likelihood of achieving water-resistant patterns by decreasing printing speed. To show the penetration of the ink through the paper, we first plotted lines on chromatography paper with different plotting speed and number of passes, and then demonstrated the cross-sections using optical microscope Fig. . Figure In addition to investigating the effect of plotting speeds, circles with different diameters (from 1.6 mm to 4 mm) were plotted to determine the resolution achievable with AxiDraw while still producing water-resistant patterns. Figure The result of the paper feeder accuracy test is obtained by plotting the pattern shown in Fig. To verify the effectiveness of the presented method for fabrication of paper-based microfluidics, a glucose assay was conducted on two different types of paper using colorimetric analysis. Glucose solutions with different concentrations  and volumes  were used to show the effect of concentration and volume on color intensity. Figure To verify the multiplexing capability of the presented fabrication method in simultaneously running multiple assays on a single device without cross-contamination, we used the plus-shaped patterns with four detection zones. Two of the detection zones were spotted with the glucose reagent and two of them were the designated controls (one with the reagent solution without potassium iodide (KI) and one with no solution). Figure 29. Patterns were plotted onto chromatography paper and delicate task wipers using permanent markers, and the ink was allowed to penetrate completely through the paper. Chromatography paper was selected because of its uniform thickness and wicking properties while delicate task wipers have a favorably high wicking speed. Based on the commercial information, the thickness of chromatography paper used here is 180 µm, which is consistent with our presented SEM images , and a colorantges Fig. . The thiAfter evaporation of the solvent, the hydrophobic resin and the colorant remain in the paper and create a hydrophobic barrier throughout the thickness of the paper. Aqueous samples are contained within the hydrophilic zones delimited by the hydrophobic barriers. To assess the water resistance of the permanent markers, aqueous dye was added to circular patterns plotted on both the delicate task wipers and chromatography paper using a desktop pen plotter. As shown in Fig. Figure To show an application of the presented fabrication method in bio-chemical analysis field, we conducted glucose assay. Results Fig.  demonstrChoosing an appropriate patterning method for mass fabrication of paper-based microfluidics requires a trade-off between cost, convenience, and resolution. We have demonstrated an inexpensive, high-throughput, simple, and rapid prototyping method for fabrication of paper-based microfluidics by using a desktop pen plotter, a customized paper feeder, and a permanent marker on delicate task wipers and chromatography paper. With the proposed technique, the fabrication cost of a 10 cm by 100 cm paper microfluidic strips is less than $1 and takes less than 15 min. To demonstrate the potential applications of this fabrication method in biological and chemical analysis, a glucose assay was performed, and the results were quantified. The ink capacity of the commercial permanent marker may be considered as a drawback of this technique regarding mass production, but that could easily be addressed by supplying the marker with a larger ink reservoir. Furthermore, an empty marker could be used for dispensing bioreagents onto the detection zones of paper microfluidics. By considering the cost and resolution offered by this fabrication method as well as ease of fabrication, this approach is highly-suitable for fabrication of paper-based microfluidics for different applications including point-of-care diagnostics, food safety monitoring, and environmental testing, especially in poor-resource areas.A desktop pen plotter, AxiDraw ; Deli markers with different colors ; Sipa markers with different colors ; Double-ended Comix markers , where the fine-tip and broad-tip markers are 0.5 and 2.0 mm in diameter, respectively; delicate task wipers, KIMTECH ; chromatography paper, Whatman No. 1 ; glucose oxidase/peroxidase reagent (G 3660), potassium iodide (793582), trehalose (PHR1344) all from Sigma- Aldrich, MO, US; pure glucose ; a microcontroller, Arduino nano .To investigate the capability of the markers to create hydrophobic barriers resisting aqueous solution, we tested various commercial brands of markers including Sipa (oil-basedmarker), Deli and Comix (permanent markers). The patterns, circles with a diameter of 4 mm, were prepared withSolidWorks and plotted by AxiDraw pen plotter on two different types of paper (chromatography paper and delicatetask wipers). The water-resistant performance of the patterns plotted by six different colors of Sipa markers,two different colors of Deli markers, and one color of double-ended Comix markers (both fine- and broad-tips)were investigated by spotting aqueous solution of yellow food dye in the center of the patterns. To increase theamount of ink diffusing through the paper, multiple passes of plotting were performed.AxiDraw is a desktop pen plotter with XY resolution of 80 steps per mm, with ±0.1 mm reproducibility, and a maximum plotting speed of 177 mm/s. The speed is adjustable and directly affects the amount of ink diffused through the paper and the feature size of the plotted patterns. A range of speeds from minimum (1%) to maximum (110%) was tested for plotting circles with diameters from 1.6 to 4 mm. Comix (both fine- and broad-tip) and black Deli markers were tested for this set of experiments as they successfully created hydrophobic barriers.To achieve a high-throughput method for fabrication of paper-based microfluidics, we developed a paper feeding mechanism synchronized with AxiDraw desktop pen plotter. We created the custom paper feeder using a feeder and its motor from an inkjet printer (HP OfficeJet 6500 A). The feeder consists of two rollers (1.6 cm in diameter) with a rubber coating to grip the paper. The rollers push the paper down onto the base to keep it inline and level during patterning. An electrical circuit consisting of an external DC power supply, a Mosfet, and a microcontroller is used to control the entire system through a PC. Using an open-loop controlling algorithm, the microcontroller controls the rotation of the motor in accordance with the pattern size and drawing speed. To activate the roller, the electrical circuit is powered by 5 volts and 0.5 amperes, provided by the external DC power supply. The roll of paper is held using a bracket Fig.  designedThe accuracy of the paper feeder was tested to assess its performance. The microcontroller was programmed to run the motor for 1 sec to unravel 10 mm of the paper roll, then stop for 10 sec to allow Axidraw to plot a 5 by 5 mm square. All squares were plotted at the same XY position by sending the plot command via the PC. A Carson eFlex digital camera  was used to capture images at a fixed position, focused on the center of the square, for analyzing the position of patterns. 120 repetitions were performed, and the images were analyzed using a MATLAB script  to measure the repeatability and accuracy of the feeder.32.To demonstrate the applicability of the proposed fabrication method for paper-based microfluidics in bioassays and verify their effectiveness, we conducted a glucose assay with different concentrations and volumes of glucose solutions (1–50 mM and 1–15 μl). This method relies on the color shift from clear to brown due to the enzymatic oxidation of iodide (I−) to iodine I2). First, glucose is oxidized to produce hydrogen peroxidase (Equation (. First, Using the fine-tip Comix marker, circular patterns with a diameter of 4 mm and plus-shaped patterns with 4 detection zones and 4 channels were plotted on both delicate task wipers and chromatography paper with speeds of 80% and 4%, respectively Fig. . To show


Several studies have estimated the associations between extreme temperatures and mortality and morbidity; however, few have investigated the attributable fraction for a wide range of temperatures on the risk of out-of-hospital cardiac arrest (OHCA). We obtained daily records of OHCA cases in the 47 Japanese prefectures between 2005 and 2014. We examined the relationship between OHCA and temperature for each prefecture using a Poisson regression model combined with a distributed lag non-linear model. The estimated prefecture-specific associations were pooled at the nationwide level using a multivariate random-effect meta-analysis. A total of 659,752 cases of OHCA of presumed-cardiac origin met the inclusion criteria. Overall, 23.93%  of OHCA was attributable to temperature. The attributable fraction to low temperatures was 23.64% (95% eCI: 19.76–25.87), whereas that of high temperatures was 0.29% (95% eCI: 0.21–0.35). The attributable fraction for OHCA was related to moderate low temperature with an overall estimate of 21.86% (95% eCI: 18.10–24.21). Extreme temperatures were responsible for a small fraction. The majority of temperature-related OHCAs were attributable to lower temperatures. The attributable risk of extremely low and high temperatures was markedly lower than that of moderate temperatures. Temperature-related mortality and morbidity is a growing public health concern2OHCA is an on-going public health issue with a high case fatality rate and associated with both patient and environmental factors. Previous studies have shown that patient characteristics such as gender, age, weight, diet, smoking, physical activity, socioeconomic status, family history of cardiac arrest, medical history of heart disease, race, and emergency medical services (EMS) are associated with incidence/survival of OHCA5679101113141516We investigated the population attributable risk of OHCA due to temperature and the relative contributions of low and high temperatures over a 10-year period in the 47 Japanese prefectures using flexible and advanced statistical approaches based on multivariate meta-regression models with time-varying distributed lag non-linear models141516171819202122A total of 1,176,351 cases of OHCA occurred between January 1, 2005 and December 31, 2014 in the 47 Japanese prefectures. Of those, we evaluated 659,752 cases of OHCA of presumed-cardiac origin that met the inclusion criteria , Table 1Tokyo Prefecture had the largest population and, thus, was selected as a representative prefecture. p = 0.324; I2 = 3.9%).The attributable risks related to low and high temperatures according to prefecture are shown in The pooled attributable risks due to temperatures stratified by gender and age are shown in The cumulative population attributable fraction related to moderate and extreme temperatures are shown in The sensitivity analysis revealed that varying the choice of model had little effect on the estimates .Our nationwide study produced several notable findings. Most importantly, our results showed that temperature accounted for a substantial fraction of OHCAs, and that most of morbidity burden was attributable to low temperatures. Specifically, most of the attributable OHCAs were caused by moderately low temperatures, whereas the contribution of extremely high and low temperatures was relatively small.We found that most of the OHCA risk attributable to temperature was related to low temperatures. Despite increased risk of OHCA due to extreme temperatures, most of OHCA burden was attributable to moderately low temperatures. Our findings are consistent with those of previous studies showing that sudden cardiac death increased in winterAn association between cardiovascular events and temperature is supported by previous studies showing that hospitalization for cardiovascular disease increases when temperatures are low282928293334Low temperatures trigger bronchoconstriction and impair the mucociliary clearance system, and other immune responses, resulting in local and systemic inflammation and increased risk for respiratory infectionsI2 index. However, it is worth noting that both are dependent on the power of the analysis, with the I2 index increasing and the p-value of the test decreasing proportionally to the precision of the estimatesA random-effect multivariate meta-analysis revealed no significant geographical heterogeneity in the temperature-OHCA relationships between prefectures. Our results are consistent with a previous meta-analysis, which indicated that latitude had little effect on cardiovascular morbidityOur findings suggest that a better understanding of the relationships between high and low temperatures and OHCA is essential for the identification of specific factors that affect susceptibility to high and low temperatures. In Japan, the demand for emergency ambulances has nearly exceeded capacity, forcing public health officials to reduce the use of ambulances and develop mitigation strategies2.5 concentration was associated with ischemic heart disease mortality and morbidityOur study had several limitations. First, we used an observational design. However, because the registration of OHCA cases was reasonably comprehensive, we are confident that there were no major selection biases. Nevertheless, we acknowledge that observational studies only partially control and adjust for the factors actually measured, and as such we could not control for unknown confounding factors or prevent bias. Second, we did not control for air pollution. A recent study found that PMIn conclusion, despite its limitations, our study showed that most of the temperature-related OHCA burden was attributable to low temperatures. The effect of extreme temperatures was substantially less than that of moderate temperatures. Our findings suggest that public health interventions to control temperature-related OHCA should take a wide range of temperatures into account.Detailed information on EMS system in Japan is published elsewhere454747Daily mean temperatures and relative humidity data collected by representative monitoring stations in each prefecture were obtained from the Japan Meteorological Agency. Daily mean temperature was selected as the main exposure index because it reflects exposure throughout the day than maximum or minimum temperatures, which occur instantaneously, and can be readily interpreted for decision-making purposes49Our study was approved by the Institutional Ethics Committee of Kyushu University Graduate School of Medical Sciences. This was an observational study using national registry data; thus, the requirement for written informed consent was waived.We conducted a two-stage time-series analysis using data from 47 Japanese prefectures to investigate the OHCA risk attributable to temperature. In the first stage, we estimated prefecture-specific temperature–OHCA relationships using a time-series regression model, which allowed for nonlinearity and delayed effects. In the second stage, a multivariate meta-regression analysis was used to pool the prefecture-specific estimates at the national level.We first examined the association between temperature and OHCA in each prefecture using a time-series quasi-Poisson regression model combined with a distributed lag non-linear model1819Associations between prefecture and temperature-induced morbidity are typically evaluated using an absolute temperature scale; however, because the range and distribution of temperatures varied across the 47 prefectures, combining temperature-OHCA curves across prefectures using non-overlapping temperature ranges was problematic. Moreover, relative temperature scales may provide a more reliable indicator of the overall effect of temperature on OHCA than absolute scales because they reflect adaptation to climate change over time and space. Thus, the associations between temperature and OHCA were evaluated using a relative temperature scale according to percentiles of the prefecture-specific mean temperature distribution.I2 indexThe estimated prefecture-specific associations were then pooled using multivariate meta-regression models obtained following reduction of the time-series regression model to estimate the non-linear temperature–OHCA relationship at the national levelFitted multivariate random-effects meta-regression models were used to estimate the best linear unbiased prediction of the overall cumulative associations between temperature and OHCA in each prefecture. The optimal linear unbiased prediction represented a trade-off between the prefecture-specific association tested by the time-series regression model and the prefecture-pooled association. This approach enabled areas with a small number of daily cases or a short time-series to use information from larger populations sharing similar characteristics14The temperature with the lowest risk of OHCA morbidity was defined as the MMT. The MMT, which corresponds to a minimum morbidity percentile (MMP) between the 1st and 99th percentiles, was derived from the best linear unbiased prediction of the overall cumulative association between temperature and OHCA in each prefecture. We used the MMT as the reference for calculating the attributable risk by re-centering the quadratic B-spline. We referred to this value as the optimal temperature. This method has been described in detail elsewhere14The total attributable number of OHCAs due to non-optimal temperature was calculated by the sum of the contributions from all days of the series. The ratio with the total number of OHCAs produces the total attributable fraction. The components attributable to low and high temperatures were calculated by accumulating the subsets corresponding to days with temperatures lower or higher than the MMT. These components were further divided into moderate and extreme components. Extremely low temperatures were defined as temperatures below the 2.5th prefecture-specific percentile of temperature, and extremely high temperatures were defined as temperatures above the 97.5th prefecture-specific percentile of temperature2414dlnm and mvmeta packages .For the sensitivity analysis, modeling selections were tested by controlling for different df for seasonal and long-term trends (6 and 10 df per year) and relative humidity in the 47 prefectures. All statistical analyses were performed with R software (ver. 3.2.5) using the How to cite this article: Onozuka, D. and Hagihara, A. Out-of-hospital cardiac arrest risk attributable to temperature in Japan. Sci. Rep.7, 39538; doi: 10.1038/srep39538 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Griffithsin (GRFT), an algae-derived lectin, is one of the most potent viral entry inhibitors discovered to date. It is currently being developed as a microbicide with broad-spectrum activity against several enveloped viruses. GRFT can inhibit human immunodeficiency virus (HIV) infection at picomolar concentrations, surpassing the ability of most anti-HIV agents. The potential to inhibit other viruses as well as parasites has also been demonstrated. Griffithsin’s antiviral activity stems from its ability to bind terminal mannoses present in high-mannose oligosaccharides and crosslink these glycans on the surface of the viral envelope glycoproteins. Here, we review structural and biochemical studies that established mode of action and facilitated construction of GRFT analogs, mechanisms that may lead to resistance, and in vitro and pre-clinical results that support the therapeutic potential of this lectin. On the other hand, nearly 29,000 cases and over 11,000 deaths were reported during the most recent Ebola outbreak that started in 2014. SARS-CoV and MERS-CoV belong to the corona virus family that includes the common cold. Outbreaks of both viruses (SARS-CoV in Asia in 2003 and MERS-CoV in Saudi Arabia in 2012) led to several thousand cases of SARS and MERS, and hundreds of deaths. Efforts to create vaccines against these viral diseases have been successful in some cases such as yellow fever, but other cases like HIV have been more challenging.A number of life-threatening human diseases are caused by viruses as diverse as human immunodeficiency virus (HIV), Ebola, yellow fever, Zika, influenza, and severe acute respiratory syndrome and Middle East respiratory syndrome corona viruses . Though these viruses are taxonomically and genetically distinct, they are all enveloped viruses and therefore possess a lipid bilayer that protects the viral capsid and genetic material that is inside the viral particle. Despite the efforts to eradicate these diseases, a number of them continue to affect us either as pandemic diseases or as outbreaks that appear from time to time. For example, according to the World Health Organization (WHO), by the end of 2015 there were nearly 37 million people infected with HIV, with two million new cases and about one million deaths occurring that year  consisting of a combination of small molecule antiretroviral drugs has progressively improved the lives of patients in some parts of the world, and effectively extends life expectancy. Antiretroviral drugs target different proteins and enzymes essential for the virus life cycle. On the other hand, antibody therapeutics are emerging as powerful alternative forms of therapy against HIV and other diseases. Such therapies depend on isolation of neutralizing monoclonal antibodies, primarily from patient sera, that will bind specifically to the envelope glycoprotein of the virus. Another way to treat viral diseases that is currently being studied is through the use of proteins capable of targeting the glycans present on the surface of the envelope glycoproteins, namely, lectins ,6. LectiGriffithsia sp. present in the NCI Natural Products Repository. Mass spectroscopic and nuclear magnetic resonance (NMR) data indicated the active compound was a protein rather than a small molecule natural product. Its sequence was determined through a combination of N-terminal Edman degradation of the intact protein and N-terminal sequencing of peptide fragments obtained from endopeptidase and cyanogen bromide treatments [Griffithsin was discovered as an anti-HIV lead by researchers at the National Cancer Institute (NCI). GRFT was isolated from a marine red alga eatments . The wilEscherichia coli (E. coli) with a polyhistidine tag on the N-terminus to facilitate purification by metal affinity chromatography [Nicotiana benthamiana using an infectious tobacco mosaic virus (TMV)-based vector has yielded gram amounts of the protein [A large-scale expression system is essential for the development of GRFT as an affordable drug. To that end GRFT has been expressed recombinantly in different organisms . It was tography ,10. Expr protein ,12. Expr protein . Purific protein , and a c protein . ImportaN-acetylglycosamine, and the disaccharides 1-6α-mannobiose and maltose have been reported [Griffithsin exists as a stable homodimer where each subunit contains 121 amino acids A. It hasreported ,16,17. Wreported   (Man9GlcNAc2) but lacks the core. In that structure, they found two of the three arms of nonamannoside bound to two of the three mannose-binding sites  ,20. To fKD values. Enthalpies among different saccharides indicated favorable electrostatic contacts. Large changes in entropy were reasoned by large displacement of water molecules from the binding sites [Isothermal titration calorimetry (ITC) showed that binding to mono- and disaccharides is exothermic and entropically favored with micromolar ng sites ,17,19. I50s) in single-round HIV infectivity assays.Using the structure and carbohydrate binding arrangement of GRFT, Micewicz et al. constructed a peptide, grifonin-1 (GRFN-1), that contained three covalently linked beta sheets and displayed three-fold symmetry . GrifoniTo enhance the ability of GRFT to inhibit HIV infection, Kagiampkis et al. created a covalently linked fusion protein composed of GRFT and the known HIV entry inhibitor C37 . C37 is Following on their work with monomeric GRFT, Moulaei and co-workers created several GRFT tandemers . The tanGRFT is an unusually stable protein, withstanding extraction procedures used in natural products’ isolation and purification. These may include exposure to organic solvents and repeated lyophilization. In addition to this physicochemical stability, its resistance to protease degradation was established in a study examining the stability of antimicrobial peptides. There, the authors showed that GRFT was resistant to digestion by eight out of nine different proteases and was degraded only by the metalloprotease elastase . In addi50 for neutralization is several orders of magnitude weaker than wild-type GRFT [9 interacting with each of the mannose-binding sites in GRFT [9 to engage all three binding sites on GRFT was further supported by studies that showed that glycopeptides that mimic Man9 required distances greater than those separating the arms of natural Man9GlcNAc2 in order to engage all three binding sites simultaneously [9 and homodimeric structure of GRFT led to bridging of viral particles and aggregation [The importance of intermolecular crosslinking by GRFT has been suggested in several publications ,20,21,26ype GRFT . Crystal in GRFT ,19. The regation .The ability of monomeric GRFT tandemers, linked together by Gly-Thr-Gly amino acid linkers, showed that two individual monomeric units were needed to recover (and improve by up to 5-fold) the potency of wild-type GRFT . Three u50) is presented in GRFT has been shown to have antiviral activity against a number of enveloped viruses as described in this section. Though the predicted number of glycans varies considerably among the viruses inhibited by GRFT , antivir+ target cells that include T cells, macrophages and dendritic cells (DC). HIV infection can occur cell-free via infection by HIV particles or by cell–cell contact between HIV-1 infected T cells or dendritic cells and non-infected CD4+ target T cells at a virological synapse [+ cells. This interaction triggers the same conformational changes needed for further co-receptor binding, followed by gp41 mediated membrane fusion.HIV can infect cluster of differentiation 4 positive (CD4) synapse . These s+ T cells [It has been shown that GRFT is capable of inhibiting in vitro infection of clade A, B and C viruses, with clade A being the least sensitive to GRFT . Additio T cells ,37. GRFT T cells . Thus, GInterestingly, even though the monomeric GRFT had comparable affinity towards nonamannoside, it showed decreased ability to bind to gp120 and even more dramatic decrease in inhibiting HIV infection in CEM-SS cells . This in50 values for GRFT against T/F viruses [N-linked glycosylation sites and the sensitivity to GRFT for T/F viruses, there seemed to be a correlation between the number and location of high-mannose glycan sites and the resistance to GRFT [50 values in the sub-nanomolar range [HIV transmission is believed to be mediated predominantly by transmitted/founder (T/F) viruses. These viruses show differential glycosylation when compared with chronic viruses. The effect of GRFT on T/F viruses has been measured and it has been found that there is a great variability on the IC viruses . Interes to GRFT . Lastly,ar range .R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA) that must be administered as a prodrug ester [GRFT has been shown to bind the HIV envelope glycoproteins thereby blocking CD4 binding as well as binding of other anti-HIV antibodies. These include the conformation-specific monoclonal antibody (mAb) 48d that binds to the CD4-induced epitope on gp120, and the carbohydrate-specific mAb 2G12 ,36. Inteug ester .It has been shown that GRFT competes with the glycan-specific antibody 2G12 , a mAb tIn contrast with the enhancement of binding to the CD4 binding site, it was shown that GRFT inhibits binding to the co-receptor binding site-specific mAb 17b without directly occluding the binding site . AlexandTo further investigate the effect of GRFT on exposing the CD4bs, Xue et al. showed that all six mannose-binding sites were needed to expose this site, as mutants lacking mannose-binding sites on one domain did not show CD4bs exposure . They fu+ T cells. It is believed that the interaction of HIV-1 with dendritic cells through the DC-SIGN receptor mediates the transfer of HIV-1 to the lymph nodes where it infects CD4+ T cells. The DC-SIGN mediated transfer of HIV to target cells involves binding of the virus to the DC-SIGN receptor, a process that occurs through interactions between high-mannose glycans on the surface of the envelope glycoprotein with DC-SIGN, followed by transfer to CD4+ target cells for infection. GRFT has been shown to inhibit both processes [Dendritic cells are antigen-presenting cells responsible for capture and degradation of pathogens that enter the mucosal environment. DCs also play an important role in viral transmission. DC cells present a DC-SIGN (dendritic cell intercellular adhesion molecule 3 (ICAM-3) grabbing non-integrin) receptor on their surface. DCs migrate to the lymph nodes where the DC-SIGN receptor mediates the transmission to naïve uninfected CD4rocesses ,41. In arocesses . Competirocesses ,40. GRFTrocesses .50 values of observed for GRFT against numerous HIV-1 subtype A and B strains, with the glycosylation of naturally-occurring viruses [Several excellent studies investigating mechanisms of GRFT resistance in HIV have been published. Not surprisingly, one strategy used by HIV against all lectins as well as glycan-targeting mAbs involves deletion of glycosylation sites . Alexand viruses . Their r viruses  on the o viruses .Oscillatoria agardhii agglutinin (OAA), GRFT-resistant HIV-1 IIIB strains were generated and resulted in deletions of glycans in positions 230, 234, 295 386 and 448, and these multiple mutations decreased GRFT susceptibility by more than 10,000 fold [In a separate study, the Morris and Moore laboratories examined the mechanisms of resistance of an HIV-1 subtype C strain to lectins including GRFT. They found that viruses cultured in the presence of increasing concentrations of GRFT evolved deletions of several glycans including those in positions 339, 230, 234, 241, 298, 392 and 448. Insertion and deletion of amino acids near the glycosylation sites in the V4 loop of gp120 that would be expected to alter glycosylation were also observed . Importa000 fold . Though A number of laboratories have studied synergistic activities between GRFT and other proteins including antibodies and lectins as well as commonly used antivirals.The monoclonal antibody 2G12 has been used in several studies because it recognizes a carbohydrate-dependent epitope comprising a cluster of high mannose glycans. These include glycans at Asn residues 332, 339 and 392, sites that are suggested to be targets of GRFT on the basis of resistance profiles. In competition enzyme-linked immunosorbent assays (ELISA), capture of GRFT-treated virus by immobilized 2G12 showed these proteins to be competitive with one another . Howeverhippeastrum hybrid (amaryllis) lectin (HHA), and galanthus nivalis lectin (GNA) in virus infectivity assays [Owing to their potency and general potential as topical microbicides, numerous studies have looked at the effects of synergy between GRFT and other lectins, including mannan-binding lectin (MVN), banana lectin (BanLec), y assays . Synergyy assays . BanLec y assays . Interesy assays . As withIt has been shown that GRFT synergizes with known HIV-1 drugs, including the nucleotide reverse transcriptase inhibitor tenofovir, the CCR5 HIV co-receptor antagonist maraviroc, the gp41 fusion inhibitor enfuvitride and the specific CXCR4 antagonist AMD3100 against using different HIV-1 clades in virus infectivity assays as well as cell–cell transmission assays ,48. ThesGRFT has been shown to inhibit infection of HCV to hepatocytes in vitro as well as cell-to-cell transmission. This has important clinical relevance because, following liver transplantation in HCV-infected patients, reinfection of the new liver graft commonly occurs ,32. Co-iCoronaviridae are enveloped positive-strand viruses that are the causative agent of severe acute respiratory syndromes (SARS). Well-known strains and outbreaks include the SARS-CoV outbreak of 2002 and the MERS-CoV outbreak of 2012. GRFT has been shown to inhibit corona virus replication and cytopathicity induced by the SARS-CoV, as well as other Coronaviridae viruses [50 in the low nanomolar range, with minimal toxicity on control cells [The  viruses ,30. In pStudies on mouse-adapted MA15 SARS-CoV showed that mice that received daily doses of intranasally administered GRFT had 100% survival rate, with no loss in weight, improved lung histopathology scores, and reduction of lung tissue virus titers, compared to the control group that experienced weight loss and had only a 30% survival rate [50 in the nanomolar range [Japanese encephalitis virus is a mosquito-borne flavivirus, similar to dengue, yellow fever and Zika. The envelope glycoprotein of JEV contains two potential glycosylation sites that are important for virus infection. It has been shown that GRFT can inhibit infection of baby hamster kidney (BHK)-21 cells with ICar range , and thaar range .50s in the nanomolar range [In in vitro studies, GRFT showed weak-to-moderate activity in blocking HSV-2 entry, yet it was capable of inhibiting cell-to-cell spread with ICar range ,42. In iar range .In vitro experiments have shown that GRFT has anti-HPV activity ranging from the high nanomolar to the low micromolar range . HPV is In a study investigating the effects of three lectins on delta and gamma retroviruses including human T cell leukemia virus, GRFT had no effect .Trichomonas vaginalis. Development of microbicide agents against HIV can benefit also the prevention against Trichomonas. GRFT binds to the surface of Trichomonas and Tritrichomonas and causes flagellated trichomonads to undergo self-aggregation and precipitation [Tritichomonas in mice reduced the recovery of parasites in mice, but it did not eliminate it [Trichomoniasis is a sexually transmitted parasitic disease, caused by pitation . Such ininate it .The cytotoxicity profile of GRFT has been studied extensively. While GRFT is by now well known to prevent infection by a number of diverse viruses, there has been no sign of cellular toxicity against a variety of cells even at concentrations as high as 500 nM . If usedStudies have shown that GRFT is stable and maintains similar anti-HIV activity after incubation for long periods of time in a variety of environments, including in the acidic (pH 4–6) cervical/vaginal lavage fluids from pig-tailed macaques . GRFT biHIV can infect PBMCs, which are often used in evaluating HIV infectivity. GRFT binds the surface of PMBCs and prevents viral replication even after washing the cells . HoweverGRFT has also been proposed to treat respiratory infections such as SARS. In such cases, the protein would be administered intranasally. Preliminary studies where GRFT was administered intranasally against SARS-CoV infected mice showed that the levels of cytokines decreased compared those infected with SARS-CoV alone . PerivasSubcutaneous injection has been tested to show the potential of GRFT to treat HCV. Subcutaneous injection of GRFT in immunodeficient transgenic mice indicated that the lectin remained bioavailable even after 18 days, with only mild transient alterations in health parameters such as body weight and morbidity scores. However, prolonged dosing had deleterious effects on the overall health of these mice . The autPharmacokinetic properties of GRFT were further tested in two rodent models. Subcutaneous injections of high amounts of GRFT (50 mg/kg) showed that GRFT persists in plasma for at least two weeks, and sera tested in HIV infectivity assays demonstrated that the GRFT in sera retains antiviral activity during this time. All animals survived the high dose GRFT treatment, and some accumulation in spleen, kidney and liver was observed . AlthougAnother study assessed the immunogenicity of HIV-1 gp120 and Gag, in the presence and absence of GRFT ,54. Immu9 oligosaccharide, leaving the third binding site available for binding a second Man9 molecule. This precise distribution of carbohydrate binding sites together with the multivalent character of GRFT arising from its homo dimeric structure leads to the formation of a complex crosslinked network, likely one of the determining factors for its picomolar activity.GRFT belongs to a group of lectins capable of inhibiting HIV infection as well as infection by other enveloped viruses. GRFT binds the terminal mannose units present on high mannose oligosaccharides present on the surface of various viral envelope glycoproteins. Structural studies by X-ray crystallography and NMR have shown that the unique structure and distribution of mannose binding sites engender GRFT with an unprecedented ability to block HIV at much lower concentrations than other anti-HIV drugs. Even though GRFT has three mannose binding sites on each subunit, it has been shown that only two of them can be simultaneously engaged by the ManGiven that the primary binding site of GRFT comprises the carbohydrates present on the surface of HIV-1 gp120, it was reasonable to evaluate the activity against other enveloped viruses. All the viruses that have been shown to be sensitive to GRFT inhibition contain moderate- to heavily-glycosylated envelopes, with a high density of high-mannose oligosaccharides present on many. The therapeutic potential of GRFT against these viruses will depend on the mode of entry of each virus. In closing, a growing body of literature has now established the remarkable potency, broad-spectrum anti-viral activity and safety profile of GRFT supporting its development as a microbicide, especially for diseases where preventative measures are unavailable.


Post-hospital discharge is a vulnerable time for recurrent illness and death among children. An intervention package consisting of (1) referrals for scheduled follow-up visits, (2) discharge counseling, and (3) simple prevention items such as soap and oral rehydration salts resulted in much higher health seeking and hospital readmissions compared with historical controls. Post-hospital discharge is a vulnerable time for recurrent illness and death among children. An intervention package consisting of (1) referrals for scheduled follow-up visits, (2) discharge counseling, and (3) simple prevention items such as soap and oral rehydration salts resulted in much higher health seeking and hospital readmissions compared with historical controls. Recurrent illness following hospital discharge is a major contributor to childhood mortality in resource-poor countries. Yet post-discharge care is largely ignored by health care workers and policy makers due to a lack of resources to identify children with recurrent illness and a lack of cohesive systems to provide care. The purpose of this proof-of-concept study was to evaluate the effectiveness of a bundle of interventions at discharge to improve health outcomes during the vulnerable post-discharge period.The study was conducted between December 2014 and April 2015. Eligible children were between ages 6 months and 5 years who were admitted with a suspected or proven infectious disease to one of two hospitals in Mbarara, Uganda. A bundle of interventions was provided at the time of discharge. This bundle included post-discharge referrals for follow-up visits and a discharge kit. The post-discharge referral was to ensure follow-up with a nearby health care provider on days 2, 7, and 14 following discharge. The discharge kit included brief educational counseling along with simple preventive items as incentives  to reinforce the education. The primary study outcome was the number of post-discharge referral visits completed. Secondary study outcomes included satisfaction with the intervention, rates of readmission after 60 days, and post-discharge mortality rates. In addition, outcomes were compared with a historical control group, enrolled using the same inclusion criteria and outcome-ascertainment methods.During the study, 216 children were admitted, of whom 14 died during hospitalization. Of the 202 children discharged, 85% completed at least 1 of the 3 follow-up referral visits, with 48% completing all 3 visits. Within 60 days after discharge, 22 children were readmitted at least once and 5 children (2.5%) died. Twelve (43%) readmissions occurred during a scheduled follow-up visit. Compared with prospectively enrolled historical controls, the post-discharge referral for follow-up increased the odds of readmission  and care sought after discharge . Overall satisfaction with the bundle of interventions was high, with most caregivers strongly agreeing that the discharge kit and post-discharge referrals improved their ability to care for their child.Interventions initiated at the time of discharge have the potential to profoundly affect the landscape of care during illness recovery and lead to significantly improved outcomes among children under 5 years of age. In resource-poor countries, in-hospital death rates for children hospitalized for a serious infection are similar to death rates in the weeks after they return home.This study builds on an earlier observational study in Uganda conducted between 2012 and 2014, in which we observed children who had been admitted to the hospital with proven or suspected infections, for the purpose of developing prediction models for post-discharge mortality.The objective of this proof-of-concept study was to determine the effectiveness of a discharge package—consisting of a discharge kit  and a post-discharge referral for scheduled follow-up visits—in improving families’ health-seeking behavior from a qualified provider, and ultimately in improving mortality during the post-discharge period.This proof-of-concept study builds upon an earlier observational study in Uganda in 2012–2014, which had the primary objective to derive models to predict post-discharge mortality.This study builds upon an earlier observational study, in which modeling predicted post-discharge mortality among children. This proof-of concept study provided a post-discharge intervention to improve mortality.This study was conducted at 2 sites—the Mbarara Regional Referral Hospital and the Holy Innocents Children’s Hospital, both in Mbarara, Uganda. The Mbarara Regional Referral Hospital is a public hospital funded by the Uganda Ministry of Health and is associated with the Mbarara University of Science and Technology Faculty of Medicine. The pediatric ward admits approximately 5,000 patients per year. Holy Innocents Children’s Hospital is a Catholic children’s hospital offering subsidized fee-for-service outpatient and inpatient care in Mbarara and admits approximately 2,500 patients annually. These study sites were chosen to reflect the relatively high proportion and use of both private and public hospitals in Uganda, and to improve external validity by comparing 2 types of institutions.The earlier observational study was approved by the institutional review boards at the University of British Columbia  and the Mbarara University of Science and Technology , the details of which have been published.Using the same criteria as the observational study,The proof-of-concept study used the same research nurses, field officers, and all equipment as in the earlier observational study. Following enrollment, a research nurse obtained and recorded clinical signs and symptoms as described in the observational study.During enrollment in the study, children received routine care according to the Uganda National Guidelines until the point of discharge. At discharge, the children received a bundle of interventions. The bundle consisted of (1) post-discharge referral for follow-up visits organized by the research nurse at the time of discharge, and (2) a discharge kit consisting of counseling and simple preventive items to reinforce the counseling.,During the discharge counseling, the research nurse provided the child’s caregiver with a paper referral form for follow-up with either a community health worker or at a nearby health center on days 2, 7, and 14 following discharge. These days were chosen as the highest-risk times during the early post-discharge period.supplementary material). Using this card, the nurse explained the child’s vulnerability during the discharge period and discussed 3 main themes of action: (1) prevention through hygiene and other health behaviors , (2) recognition of signs of early illness recurrence, and (3) prompt care sought at a health center or from a community health worker. In our earlier research, these themes featured prominently in the children who died following discharge.The caregivers also received a discharge kit, which included brief educational counseling paired with simple preventive items as incentives to reinforce the education. The educational counseling consisted of a storyboard-style, laminated card written in the local language  tool hosted at the Child and Family Research Institute in Vancouver, Canada.The primary study outcome was the proportion of children who successfully completed at least 1 post-discharge referral for a follow-up visit at a health center or with a community health worker. Secondary study outcomes included caregiver satisfaction with the interventions  and comparisons in post-discharge mortality, readmission, and care sought between this study and the earlier observational study.We performed descriptive analyses of the primary outcome and of caregiver satisfaction. Using logistic regression, we also performed comparative analyses between the earlier observational study and this study. In this analysis, the outcomes of readmission and post-discharge health seeking were adjusted for both site of enrollment and the post-discharge mortality risk score. The post-discharge mortality risk score is a 5-item composite score including mid-upper arm circumference, oxygen saturation, time since most recent hospitalization, HIV status, and coma score. We also conducted a secondary univariate logistic regression analysis examining factors associated with completing post-discharge referrals. For the secondary analysis, a sample of 200 children would provide 80% power to detect a 10% absolute difference in health seeking compared with the historical cohort, at an alpha of .05. All analyses were conducted in SAS 9.4 .z score less than -2). One hundred and four (48%) were diagnosed with pneumonia, while 45 (20%) were diagnosed with malaria and 17 (8%) with diarrhea. The rate of in-hospital mortality was 6.5%.A total of 216 children were enrolled in the study. An additional 93 children were screened but excluded from the study, mostly because they presented with a non-infectious illness . The medThe number of children who survived to discharge was 202 (93.5%). Of these, 170 (84%) completed at least 1 scheduled post-discharge follow-up visit, 143 (71%) completed 2 follow-up visits, and 96 (48%) completed all 3 follow-up visits. Among those children who did not complete all 3 follow-up visits, the 3 most commonly reported reasons for non-completion were (1) health system factors, such as a closed health center; (2) family factors, such as lack of transportation or high cost; and (3) the child’s family did not consider the visit important . For the84% of children surviving to discharge attended at least 1 follow-up referral visit after discharge, but less than half completed all 3.z score, who had used a mosquito net consistently, and who had a length of stay of less than 5 days during the initial admission. Age, sex, household crowding, sibling deaths, and age of the child’s mother were not associated with follow-up success  children were readmitted at least once, for a total of 28 admissions . Of thosWithin 60 days after discharge, 11% of children were readmitted.Approximately 60 days after discharge, field officers administered a short survey with 5 questions for caregivers to solicit feedback on the bundle of interventions . Overall75% of caregivers were very satisfied with the interventions.In the earlier observational study, 1,307 children were enrolled using the same enrollment criteria as this study. Of these, 65 died during hospitalization (5%), resulting in 1,242 live discharges . During Compared with the earlier observational cohort study, we found a 25% lower odds of death at 60 days, a nearly 2-fold higher adjusted odds of readmission, and a 14-fold higher adjusted odds of seeking post-discharge care. ,The absolute increase in care seeking post-discharge increased from approximately 30% in the earlier observational study (with no intervention) to nearly 90% in the current study . This indicates that less than 2 children would need to receive this intervention in order for 1 additional child to receive in-person follow-up care who otherwise would not have received follow-up care after discharge. The characteristics of these 2 samples were similar. However, the earlier observational cohort had a higher proportion of patients with severe malnutrition. The interventional cohort had a substantially higher proportion of pneumonia and lower proportion of malaria (and thus a lower mean oxygen saturation). The proportion of children with HIV and of those with an abnormal Blantyre Coma Scale score was slightly higher in the interventional cohort . When apThis proof-of-concept study evaluated the effectiveness of using a discharge bundle, consisting of a discharge kit (education and preventive items as incentives) and post-discharge referrals for follow-up visits, in a hospital environment in southwestern Uganda to improve health-seeking behavior and post-discharge mortality. Results showed that this intervention was both feasible and effective. After administering this intervention to the caregivers of some 200 children, we found that 89% of children successfully achieved at least 1 post-discharge follow-up visit, with nearly 50% completing all 3 follow-up visits. The observed falling compliance in referral completion, however, and the fact that 40% of missed visits were due to circumstances that appeared to be beyond the control of the caregiver, suggest that 2 visits may be more practical in bringing this type of intervention to scale.Two, instead of three, follow-up visits post-discharge may be more practical in bringing this type of intervention to scale.,Post-discharge care was community focused, with referrals being directed to community health workers and nearby health centers. Three-quarters of caregivers reported that these visits were neither inconvenient nor difficult. Most strongly agreed that the education and referral were important components of care for their child’s recovery. Compared with prospective observational data collected before this study, this package of interventions was associated with increased hospitalizations , with over 40% of readmissions being directly linked to a referral visit. Overall health seeking from a community health worker or a health center post-discharge was only 30% in the previous observational study with no intervention compared with nearly 90% in the current study that included the package of interventions to improve health seeking. This reflects an important achievement given that modeling in the observational study predicted that more than 1 of every 30 children discharged is likely to die within the first 2 months and that health-seeking behavior is poor for the most common causes of childhood death.Post-discharge mortality is a neglected but important issue in the field of pediatric global health. Prior research has shown that, in some areas, mortality after discharge exceeds mortality in the hospital.Post-discharge mortality is a neglected but important issue in the field of pediatric global health.Although post-discharge care should be an important component in the care of all discharged children, limited resources and an already strained health system are likely to be major barriers in its implementation. The current context among hospitals in Uganda and elsewhere is that the limited post-discharge care that does occur focuses primarily on the ongoing treatment of specific diseases such as HIV and tuberculosis. We are not aware of any hospitals in resource-poor settings that have adopted general discharge policies. In addition to a lack of robust policies for post-discharge care, the larger issue is the availability of resources, which is a major limitation to the scalability of effective post-discharge care. At the community level, the capacity to complete follow-up visits is sufficient and does appear to exist in most areas, but the hospital resources to assess children for risk and to implement the referral process are likely to be insufficient. We propose the establishment of discharge nurses to oversee this role, as has been suggested for improving post-stroke outcomes in sub-Saharan Africa.We propose that discharge nurses should assess children for risk and implement a referral process to improve health outcomes after discharge.,The identification of the most vulnerable children presents an important strategy to improve the cost-effectiveness of post-discharge care, as limited resources can be used to target high-risk children. Our group recently derived a clinical prediction tool that uses 5 easily collected variables to identify such children.Prior research has shown that a significant proportion of child deaths occur outside of health facilities.This proof-of-concept study is subject to several important limitations. First, the small sample size limits the ability of this study to make robust comparisons with the earlier observational study. Although comparisons of study outcomes such as health seeking and readmission provide some insight into the potential value of a post-discharge intervention, a primary study outcome based on mortality is preferable and will be a primary component of future research. The results of this study, however, serve an important role in guiding the design and sample size calculations for subsequent research, as does the feedback provided by the participants in the study. A further limitation of this study, and similar studies, is that the incorporation of post-discharge follow-up in a research context may not be easily replicated in a non-research context. The discharge education given to caregivers, highlighting the vulnerability of their children during the post-discharge period, likely played an important role in motivating them to sacrifice time and money to complete the follow-up referral visits. Follow-up interventions, therefore, must be strongly linked to education at the time of discharge. A final limitation of this study is the relatively narrow age range (6 months to 5 years) of children. The reason this age range was chosen was based on design considerations for the earlier observational study, which we used to develop the prediction model. Our research group is currently working toward the expansion of these prediction models to eventually include both younger (<6 months) and older (>5 years) children. Finally, because this intervention consisted of a bundle of interventions, it is impossible to identify which components were critical in the results that were observed. The simple preventive items that complemented the education  were relatively expensive (approximately US$6.50) and may impact scalability. However, it is unlikely that these particular incentives are required in order to observe these benefits. The mosquito net was the most expensive item in the bundle (about US$5.75); it could easily be replaced with a less expensive item that complements the education while still providing value to the caregiver, and could serve as a means to reinforce the instructions provided by the discharge nurse.In conclusion, we found that a simple bundle of interventions at discharge, including brief educational counseling and a post-discharge referral, can improve post-discharge care and outcomes among children discharged from the hospital. New research is currently being planned to establish the benefit of a bundle of interventions at discharge to improve post-discharge mortality and will explore bringing such interventions to scale in Uganda.