Deep brain stimulation (DBS) of the nucleus accumbens (NAc) is a potential remedial therapy for drug craving and relapse, but the mechanism is poorly understood. We investigated changes in neurotransmitter levels during high frequency stimulation (HFS) of the unilateral NAc on morphine-induced rats. Sixty adult Wistar rats were randomized into five groups: the control group , the morphine-only group (systematic administration of morphine without electrode implantation), the morphine-sham-stimulation group (systematic administration of morphine with electrode implantation but not given stimulation), the morphine-stimulation group (systematic administration of morphine with electrode implantation and stimulation) and the saline-stimulation group . The stimulation electrode was stereotaxically implanted into the core of unilateral NAc and microdialysis probes were unilaterally lowered into the ipsilateral ventral tegmental area (VTA), NAc, and ventral pallidum (VP). Samples from microdialysis probes in the ipsilateral VTA, NAc, and VP were analyzed for glutamate (Glu) and γ-aminobutyric acid (GABA) by high-performance liquid chromatography (HPLC). The levels of Glu were increased in the ipsilateral NAc and VP of morphine-only group versus control group, whereas Glu levels were not significantly changed in the ipsilateral VTA. Furthermore, the levels of GABA decreased significantly in the ipsilateral NAc, VP, and VTA of morphine-only group when compared with control group. The profiles of increased Glu and reduced GABA in morphine-induced rats suggest that the presence of increased excitatory neurotransmission in these brain regions. The concentrations of the Glu significantly decreased while the levels of GABA increased in ipsilateral VTA, NAc, and VP in the morphine-stimulation group compared with the morphine-only group. No significant changes were seen in the morphine-sham stimulation group compared with the morphine-only group. These findings indicated that unilateral NAc stimulation inhibits the morphine-induced rats associated hyperactivation of excitatory neurotransmission in the mesocorticolimbic reward circuit. However, this class of psychoactive agents can also elicit intense euphoric effects followed by feelings of well-being in the user, particularly when taken in high doses, and this can lead to drug abuse and ultimately addiction Parkinson’s symptoms through the activation of GABAergic neurotransmission and increased inhibitory neurotransmitter GABA release, and consequently attenuate dopamine release Nonetheless, the mechanism underlying NAc-DBS treatment of addiction remains unclear. It was reported that addiction may be related to an imbalance between the excitatory glutamate (Glu) and inhibitory γ-aminobutyric acid (GABA) neurotransmitters The conditioned place preference (CPP) is one of the most established experimental protocols which used for measuring drug reward in laboratory The present study aimed to evaluate the effects of NAc-DBS on changes of Glu and GABA levels in NAc, VTA, and VP of morphine-induced addiction rats produced by systemic morphine administration. Intracerebral microdialysis was used to monitor the extracellular concentrations of Glu and GABA in the ipsilateral NAc, VTA, and VP.Sixty adult male Wistar rats weighing 280–320 g were provided by the Experimental Animal Center of Military Medical Sciences, Beijing, China. The animals were individually housed in cages  with free access to food and water. All experiments were performed during the light phase of the cycle. These were randomized into five groups, all of which had intraperitoneal injections: (1) the control group (n = 12), which underwent administration of saline; (2) the morphine-only group (n = 12), which underwent administration of morphine without electrode implantation; (3) the morphine-sham-stimulation group (n = 12), which underwent administration of morphine with electrode implantation but not given stimulation; (4) the morphine-stimulation group (n = 12), which underwent administration of morphine with electrode implantation and HFS stimulation, (5) the saline-stimulation-group (n = 12),which underwent administration of saline with electrode implantation and stimulation. The experimental procedure is shown in Rats in the morphine-only, the morphine-sham-stimulation and the morphine-stimulation groups received intraperitoneal injections of morphine hydrochloride  at increasing doses  and placed in a stereotaxic apparatus. A microdialysis guide cannula with a stylet  was implanted. The concentric bipolar stimulation electrodes  were glued to the guide cannula with a specially made device, and the electrode tips were 2 mm below the end of the guide cannula. Four cannulas were implanted in each rat: one unilaterally in the core of the NAc ; one within the ipsilateral core of NAc ; one within the ipsilateral VTA ; and one within the ipsilateral VP  according to the atlas of Paxinos and Watson CPP apparatus  were three-chambered shuttle boxes comprising a small central compartment (10×10 cm) where rats were placed at the start of a test session, and two larger compartments (40×40 cm), one with horizontal black and white stripes and one with vertical black and white stripes. Floors were made of stainless steel sheeting with punched-out shapes resulting in distinct textures . Removable partitions allowed the boxes to be used either to restrict the rats to a particular compartment for conditioning or to allow the rats to be ‘free-to-explore’ during a test session. Experiments were performed between 8∶00 am and 6∶00 pm under dim white light (light intensity approximately 15l ux). During all test sessions, the time each rat spent in each compartment was recorded using EthoVision XT  tracking software.Conditioned place preference consisted of a 14-day schedule with three phases: pre-conditioning, conditioning and post-conditioning. The compartment occupied for the shorter time was designated as the drug-paired compartment. Previous work in our laboratory indicated that naïve rats tend to display a preference for the black one. Therefore the white chamber was designated as the drug-paired compartment that is the site where morphine would be administered.Pre-conditioning phase: during this phase, each animal from all groups was placed in compartment with the guillotine door removed to allow access apparatus for 900 sec each day for three consecutive days. On day 3, the time rats spent in each compartment was recorded.Conditioning phase: Conditioning sessions were conducted from day 4 to day 13 with each group receiving 6 hourly alternating injections of either saline or morphine. Each conditioning session was 30 minutes in duration, once daily. From 8∶00 to 12∶00 on the first training day, rats in each group were injected with saline and immediately confined to the black compartment (not drug-paired side) of the shuttle box; from 14∶00 to 18∶00 the rats were confined to the white compartment (drug-paired side) after the saline was administered to the control and saline-stimulation group and morphine to the morphine-only group, morphine-stimulation group and morphine-sham-stimulation group. On the second day, the procedure was performed in reverse order, that is, from 8∶00 to 12:00 the rats were confined to the white one after the saline was administered to the control and saline-stimulation group and morphine to the morphine-only group, morphine-stimulation group and morphine-sham-stimulation group; from 14∶00 to 18∶00 the rats in each group were injected with saline and confined to the black one.Post-conditioning or testing phase. This phase was carried out 1 day after the last training in a morphine-free state. On the test day, the removable wall was raised and the rat could access the entire apparatus for 900 seconds. Conditioned place preference was indicated by a preference score, defined as the time spent in the drug-paired side minus the time spent in the saline-paired chamber.2, 1.2 mM; CaCl2, 1.2 mM; pH 7.4) at a flow rate of 2 ml/min through a microsyringe pump . The dialysis tubing was connected to a liquid swivel for collection from the lightly anesthetized animals. A 2-h stabilization period was allowed before the collection of dialysates for analysis, and dialysate fractions were then collected at 30 min intervals. The three fractions were collected during stimulation. Dialysates were automatically collected with a refrigerated autosampler and stored at –80°C until analysis.One day after the post-conditioning phase, each rat was lightly anesthetized with pentobarbital and the three guide stylets were replaced with the microdialysis probes leaving the stimulation electrodes in place . The probes were perfused with artificial cerebrospinal fluid  using a refrigerated autoinjector. The mobile phase consisted of NaH2PO4  with 20% methanol at a flow rate of 1 ml/min delivered by a Waters pump. Glutamate and GABA were identified through their migration time and spike profiles. Peaks values were normalized to an internal standard curve for fluorescein and quantified by comparison with an external standard curve  (Neurotransmitter (Glu and GABA) levels in the dialysate samples were determined by high-performance liquid chromatography (HPLC) with fluorometric detection. Briefly, samples or standards were derivatized with O-phthaldialdehyde; 20 µl of the resulting mixture was automatically loaded onto a Novapark CEMPOWER) . The valAfter completion of all experiments, the rats were anesthetized with urethane (1 g/kg) and perfused transcardiac with 0.1 M phosphate-buffered saline followed by 4% paraformaldehyde. Brains were removed and coronal sections (30 µm) through the target were prepared and processed by hematoxylin and eosin (HE) and cresol violet staining to confirm the location of the stimulating electrode. Only rats with electrode and probe placements in the areas of interest were included in subsequent data analysis.post hoc comparisons using SPSS 13.0 analytical software for Windows . A P value of less than 0.05 was considered statistically significant.Results were expressed as means ±SEM. Repeated measures were used to determine whether the mean values of Glu and GABA at the indicated time during the unilateral NAc-DBS differed significantly from basal values. Statistical analysis employed one-way analysis of variance (ANOVA) followed by Tukey test for multiple  = 0.2134; P>0.05;   = 396.1, P<0.05). However exposure to 130 Hz high frequency stimulation was not associated with a significant increase in the time spent in the drug-paired sides in the morphine-stimulation group (−403.28±53.25 seconds) and the saline-stimulation group (−415.04±57.2 seconds) compared with the control group (−422.94±59.6 seconds)  = 396.1, P<0.05). Our data demonstrated that 130-Hz electrical stimulation of NAc significantly blocks morphine-induced CPP.At baseline (pre-conditioning), mice in each group spent a longer time on the black sides of the chamber than the white sides of the chamber (F P<0.05). No significant differences were observed in the levels of Glu in the VTA of the morphine-only, the morphine-sham stimulation group and the control groups (P>0.05). In the morphine-sham-stimulation group, no significant differences in the extracellular concentrations of Glu in NAc, VP and VTA were found compared with the morphine-only group (P>0.05). However, we observed that significant decline in the levels of Glu in NAc, VP and VTA in the morphine-stimulation group when compared with the morphine-only, morphine-sham-stimulation, saline-stimulation and control groups (P<0.05) (Significant increases in the levels of Glu in NAc and VP of the morphine-only groups and morphine-sham-stimulation group were observed when compared to the control ((P<0.05) .P<0.05). In the morphine-sham-stimulation group, we did not find statistical significance in the extracellular concentrations of GABA in NAc, VP and VTA, respectively, compared with the morphine group (P>0.05). However, we observed that significant increase in the levels of GABA in NAc, VP and VTA, respectively in the stimulation group when compared with the morphine, morphine-sham stimulation, saline-stimulation and control groups (P<0.05)  .In total, sixty animals were used. At the end of all behavioral experiments, the location of the cannula was examined histologically. Histological analysis demonstrated that the location of the electrode tips showed an identical pattern of distribution within or close to NAc, VP, and VTA in 56 animals. Another 4 animals were not included in the statistical analyses due to the mislocation or misplacement of cannulae. In addition, coronal brain sections indicated no significant cell necrosis or neuronal loss in the NAc surrounding the lead contact in the the morphine-stimulation group .To address the mechanism underlying the use of DBS as a potential therapy for morphine induced rats, we investigated the DBS effects of NAc on GABAergic and glutamatergic activity within the mesocorticolimbic system. Successful induction of a state of morphine-conditioned preference in rats treated with systematic administration of morphine was confirmed in a CPP test. Using microdialysis probes implanted into the ipsilateral NAc, VTA, and VP, we then measured levels of the neurotransmitters Glu and GABA.The study showed that morphine-conditioned status was accompanied by a characteristic pattern of changes in the brain tissue concentrations of these neurotransmitters. The levels of Glu increased in the NAc and VP of morphine-only group compared to that of the control group, although the level of Glu in the VTA did not change significantly. However, levels of GABA decreased significantly in all three regions  of morphine-only group versus control group. These results demonstrated that rats with morphine-conditioned status displayed neurotransmitter changes associated with heightened activation of excitatory pathways in mesocorticolimbic structures.The study also demonstrated that high-frequency electrical stimulation of the unilateral NAc could substantially reverse these changes. Compared to the morphine group, the levels of Glu decreased significantly in NAc, VTA, and VP, whereas GABA levels increased significantly in all three regions in the morphine-stimulation group. The result suggested that these changes could underlie the utility of DBS in the treatment of morphine-induced rats.Previous studies indicated that the effects of NAc stimulation on morphine-induced addiction were frequency-dependent. High-frequency stimulation  of the NAc was more effective than low-frequency stimulation  in treating morphine induced rats An imbalance between GABA and Glu neurotransmission was hypothesized to produce neuronal hyperexcitability in addiction, and it was previously suggested that increased levels of extracellular Glu and reduced levels of extracellular GABA are key factors in drug addiction Glu is the most prominent excitatory neurotransmitter in the brain. Following release into the synaptic cleft, Glu binds to and activates NMDA- and/or AMPA-type glutamate receptors, leading to onward propagation of action potentials. It was thought that excess glutamatergic neuronal activity may underlie drug addiction et al. demonstrated that GABA levels fell after morphine administration directly into VTA via a dialysis probe Our study also showed that morphine-addiction status in rats was accompanied by a significant decrease of extracellular GABA levels in NAc, VP, and VTA. GABA is the most important inhibitory neurotransmitter in the brain. According to the prevailing hypothesis that dopamine released in the NAc inhibits GABAergic medium spiny neurons projecting to VP, this process is thought to represent a final common path for drug reward It was reported that inhibiting extracellular Glu release or NMDA receptor expression in the NAc of morphine-treated rats, or increasing GABA concentrations in VTA or VP, could reduce opioid dependence NAc stimulation as a means to control drug addiction has shown promising results in both animal models and clinical trials. The reported low incidence of adverse events with this procedure is also encouraging. However, the mechanism by which NAc electrical stimulation might provide a treatment for drug addiction is not fully understood. Given that drug addiction in several models has been linked to increased mesolimbic activation, it is notable that, in morphine-CPP rats, unilateral NAc stimulation led to significant reductions in extracellular ipsilateral levels of Glu in NAc, VP, and VTA. Concomitantly, stimulation led to a marked rise in extracellular GABA concentrations in all three regions in our study. An obvious interpretation is that NAc stimulation dampens mesocorticolimbic hyperactivation in morphine-conditioned rats. Indeed, it seems likely that effects on neurotransmitter release may be one mechanism underlying the addiction-aborting effects of NAc stimulation.However, it has been speculated that electrical stimulation of the NAc might exert its effects by inducing lesions at the point of implantation that are provoked by the implantation of the electrode. However, we controlled for this in our study by employing an implanted electrode without electrical stimulation. The presence of an electrode implanted into NAc failed to change extracellular levels of either neurotransmitter. In addition, coronal brain sections indicated no significant cell necrosis or neuronal loss in the NAc around the lead contact. These findings, consistent with experiment Earlier studies showed that HFS can suppress somatic neuronal activity in vitro stimulation affects only local neurons, and not more distal neurons Overall, HFS of NAc is thought to inhibit the activity of NAc neurons, and this could underlie the efficacy of NAc stimulation in alleviating drug addiction Microdialysis has been widely used to sample GABA and Glu from brain. It has, however, been pointed out that there may be problems in the detecting subtle changes in amino acid levels with in vivo microdialysis technique, because these differences could be due to differences in the efficiency of probe recovery between these groups. Given there are limitations to the microdialysis technique, the present results of measurements of Glu and GABA should be interpreted with caution.In conclusion, morphine-induced CPP in rats was accompanied by increased Glu and reduced GABA levels in mesocorticolimbic regions, but NAc stimulation led to reversal, with a significant decline in Glu levels and increase in GABA levels, that are likely to reflect inhibition of the mesocorticolimbic reward circuit. These findings provided new insights into the mechanism that underlie the efficacy of deep brain stimulation in the treatment of drug addiction.Figure S1Diagram of the experimental procedure.(TIF)Click here for additional data file.


The relationship between work-related stress and alcohol intake is uncertain. In order to add to the thus far inconsistent evidence from relatively small studies, we conducted individual-participant meta-analyses of the association between work-related stress  and alcohol intake.We analysed cross-sectional data from 12 European studies (n = 142 140) and longitudinal data from four studies (n = 48 646). Job strain and alcohol intake were self-reported. Job strain was analysed as a binary variable (strain vs. no strain). Alcohol intake was harmonised into the following categories: none, moderate , intermediate  and heavy . Cross-sectional associations were modelled using logistic regression and the results pooled in random effects meta-analyses. Longitudinal associations were examined using mixed effects logistic and modified Poisson regression. Compared to moderate drinkers, non-drinkers and (random effects odds ratio (OR): 1.10, 95% CI: 1.05, 1.14) and heavy drinkers  had higher odds of job strain. Intermediate drinkers, on the other hand, had lower odds of job strain . We found no clear evidence for longitudinal associations between job strain and alcohol intake.Our findings suggest that compared to moderate drinkers, non-drinkers and heavy drinkers are more likely and intermediate drinkers less likely to report work-related stress. It has been hypothesised that stress in general and work-related stress in particular influence people's alcohol drinking habits and that work-related stress is associated with heavy drinking To date, the findings from epidemiological studies of the associations between alcohol intake and work-related stress have differed in direction and magnitude in subgroups of participants In order to better understand the relationship between alcohol drinking and stress at work, we undertook individual-participant meta-analyses of the association between work-related stress and alcohol intake. We used a large set of data pooled from 12 independent European studies with a common measure of work stress, job strain We conducted individual-level meta-analyses using pooled data from 12 independent studies conducted between 1985 and 2008 in Belgium, Denmark, Finland, France, Germany, the Netherlands, Sweden and the UK. The studies are a part of the “Individual-participant-data Meta-analysis of Working Populations” (IPD-Work) Consortium, which was established at the annual Four Centers Meeting in London, November 8, 2008. A pre-defined two-stage data acquisition protocol was used. The first stage involved the acquisition and harmonisation of baseline data on work stress as well as socio-demographic and lifestyle factors. The second stage involves the acquisition and analysis of disease outcomes. Our meta-analyses were based on the first stage cross-sectional data and were thus conducted before any linkage to disease data.Details of the design and participants of the studies included in our meta-analyses have been published previously We estimated cross-sectional associations between alcohol intake and work stress in 12 studies comprising 142 140 individuals (mean age: 44.0 years). In addition, the associations of alcohol intake and job strain in socio-demographic subgroups were investigated in studies in which we had access to individual-level data , intermediate drinkers  and heavy drinkers . The lower limit for intermediate drinking is the upper limit of healthy alcohol consumption according to public health guidelines in many countries Work stress was operationalised as job strain and defined as high job demands and low job control Information on sex and age was obtained from population registries or baseline interview with the participant  or from a participant-completed questionnaire . Sex was modelled as a binary and age as a continuous variable (years). Socioeconomic position was defined based on occupational title obtained from employers' or other registries  or participant-completed questionnaires . In HeSSup socioeconomic position was defined based on the participant's self-reported highest educational qualification. Socioeconomic position was categorised in all studies as low , intermediate  or high . Participants who were self-employed or who had missing data on job title were included in the analyses in the “other” socioeconomic position category .We used individual-level data provided by the investigators in Belstress, FPS, Gazel, HeSSup, HNR, Whitehall II, WOLF Norrland and WOLF Stockholm studies. Some groups of investigators  undertook the statistical analyses themselves according to instructions from the IPD-Work analytical team and provided the IPD-Work team with the study-specific results.2 statistic, which indicates the proportion of the total variation in the estimates that is due to between-studies variation For our main analyses, we used a two-stage meta-analytical approach because we had access to individual-level data from eight studies and aggregate data from four studies . Logistic regression models were used to estimate study-specific associations between alcohol intake and job strain alcohol intake and the resulting estimates and their standard errors were pooled using fixed effect and random effects meta-analyses All meta-analyses and statistical analyses in the pooled individual-level data were performed using Stata SE 11.0 . In POLS the study-specific analyses were undertaken by the study team using SPSS 17  and in, DWECS, IPAW and PUMA using SAS 9 .The characteristics of participants included in our analyses are shown in 2∶61.9%). The study-specific effect estimates are shown in A meta-analysis of alcohol intake and job strain is shown in We investigated access to individual-level data in the IPD-Work Consortium as a possible source of heterogeneity by stratifying our meta-analyses by the availability of individual-level data . OverallThe findings from the meta-analysis stratified by demographic covariates  and based on individual-level data are shown in Longitudinal analyses were based on pooled data with repeated measurements from Belstress, FPS, HeSSup and Whitehall II. The longitudinal associations between job strain at baseline and taking up excessive drinking (i.e. becoming an intermediate or heavy drinker) by follow-up among baseline non-drinkers and moderate drinkers are presented in In our pooled analyses of data from 12 European studies, individuals who reported abstaining from alcohol were 10% more likely to experience job strain than those who reported drinking moderate amounts. Compared to moderate drinkers, intermediate drinkers were 8% less likely and heavy drinkers were 12% more likely to report job strain, although the latter association was imprecisely estimated.Our finding that non-drinkers were more likely to report job strain than moderate drinkers is difficult to interpret because the non-drinkers are a heterogeneous group. In addition to lifelong abstainers who choose not to drink alcohol for religious or conscience reasons, this group is likely to include individuals who do not drink alcohol because of previous alcohol addiction or ill health, both of which may also be associated with work-related stress. Further research would be needed to examine the role of these factors. There are several biological mechanisms that could explain the observed lower odds of job strain in intermediate drinkers  when compared to moderate drinkers. Ethanol can produce positive mood states with stress-relieving effects through the activation of the endocannabinoid system CB1 receptors and µ/ð-opioid receptors The strengths of our investigation were that it was based on a large set of individual-level data, and as far as we are aware, is the largest study of alcohol intake and work stress to date. Thus, we were able to provide more precise estimates of the association between job strain and alcohol intake than has been possible before Our findings suggest that compared to moderate drinkers, non-drinkers and heavy drinkers are more likely to report work stress. Intermediate drinkers  were less likely to report work stress than moderate drinkers. We found no clear evidence for longitudinal associations between self-reported alcohol intake and work stress.Figure S1Studies and participants included in the analyses.(DOC)Click here for additional data file.Figure S2Associations of alcohol intake and job strain .(DOC)Click here for additional data file.Figure S3Associations of alcohol use and job strain, stratified by the availability of individual-level data .(DOC)Click here for additional data file.Table S11.Associations of alcohol intake and work job strain in demographic subgroups(DOC)Click here for additional data file.Appendix S1Study design and recruitment of participants in the 12 European studies included in our individual-participant meta-analyses of alcohol intake and work stress.(DOC)Click here for additional data file.


Lycosa carolinensis). One side of the α-helix has mostly hydrophilic and the other mainly hydrophobic amino acid residues. The positive charge of the hydrophilic side interacts with negatively charged prokaryotic membranes and the hydrophobic side associates with the membrane lipid bilayer to permeabilize it. Because the surface of the exoskeleton, or cuticle, of an insect is highly hydrophobic, to repel water and dirt, it would be expected that amphipathic compounds could permeabilize it. Mutagenized lycotoxin 1 peptides were produced and expressed in yeast cultures that were fed to fall armyworm (Spodoptera frugiperda) larvae to identify the most lethal mutants. Transgenic expression of spider venom toxins such as lycotoxin-1 in plants could provide durable insect resistance.Several important areas of interest intersect in a class of peptides characterized by their highly cationic and partly hydrophobic structure. These molecules have been called cell-penetrating peptides (CPPs) because they possess the ability to translocate across cell membranes. This ability makes these peptides attractive candidates for delivery of therapeutic compounds, especially to the interior of cells. Compounds with characteristics similar to CPPs and that, in addition, have antimicrobial properties are being investigated as antibiotics with a reduced risk of causing resistance. These CPP-like membrane-acting antimicrobial peptides (MAMPs) are α-helical amphipathic peptides that interact with and perturb cell membranes to produce their antimicrobial effects. One source of MAMPs is spider venom. Because these compounds are toxic to insects, they also show promise for development as biological agents for control of insecticide-resistant agricultural pests. Spider venom is a potential source of novel insect-specific peptide toxins. One example is the small amphipathic α-helical peptide lycotoxin-1 (Lyt-1 or LCTX) from the wolf spider ( The cell membrane is the barrier that protects living cells from the surrounding environment, generally allowing the movement of only small molecules into the cell. The hydrophobic nature of cell membranes makes them impenetrable to most peptides, proteins, and oligonucleotides. However, it was eventually observed that certain peptides had the ability to pass through cell membranes. These molecules have been called cell-penetrating peptides (CPPs). In general, CPPs are short (less than 35 amino acid residues), water-soluble and partly hydrophobic, and/or polybasic peptides with a net positive charge at physiological pH. Their ability to translocate across cell membranes is attributed to their highly cationic and partly hydrophobic structure. This class of peptides has been extensively studied for several decades, but the mechanism(s) by which CPPs enter the cells is not completely understood . AnotherLycosa carolinensis). Lycotoxin‑1 appears to function as a pore former to increase membrane permeability and to bring about lysis of cells by reducing ion and voltage gradients across membranes [Spider venom is injected into insect prey to subdue it, and is a potential source of novel MAMP insect-specific peptide toxins . An examembranes ,8. The aembranes . It woulembranes .Spodoptera frugiperda) larvae to identify the mutant toxins with greatest lethality. The most lethal mutations appeared to increase the ability of the toxin α-helix to interact with insect cell membranes and disrupt the membranes by forming pores [The potential use of lycotoxin-1 as a bioinsecticide was investigated by producing recombinant mutant lycotoxin-1 peptides via an amino acid scanning mutagenesis strategy and screened in high throughput for ability to kill fall armyworms, a significant cause of damage to corn and other crops in the United States. Yeast cultures expressing the recombinant peptide toxins were fed to fall armyworm  discovered three years later. They are the most extensively studied of all CPPs [Bombyx miori (Lepidoptera: Bombycidae). These results offer the possibility of effective oral delivery of bioinsecticidal molecules to targets located both within the insect gut epithelium and behind the gut barrier, in the hemocoel compartment, provided cargo molecules selectively acting on the pest species to be controlled are available [Many drugs are large hydrophilic molecules showing major limitations for their penetration through the cell membrane. A group of short peptides designated CPPs have been discovered that may serve as delivery vectors for large molecules, for example, therapeutic compounds ,12. The eceptors . The firvailable . During vailable . The corvailable .Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis. Attention has turned to the CPP-like MAMPs as compounds that can be used against drug-resistant microbes. MAMPs are active against a wide range of Gram-negative and Gram-positive bacteria, including drug-resistant strains. Their mechanism of interaction with cell membranes does not involve specific protein binding sites, which reduces the likelihood of inducing drug resistance. Studies are underway to understand the structure-function relationship of these peptides and develop them into useful antibiotic drugs. These studies have suggested that both pore-forming and non-pore models can be used to describe the function of MAMPs [The development of drug-resistant microbes is an increasingly serious public health problem worldwide. Recently widespread antibiotic resistance has emerged in clinically important bacterial pathogens such as of MAMPs . It is bof MAMPs ,6.Bacillus thuringiensis (Bt) crystal proteins are examples of insecticidal proteins that have been developed commercially. They are highly effective against a targeted range of species when used as topical pesticides [It is estimated that insect pests destroy approximately 14% of the World’s crop production . In manysticides ,19 and msticides ,21,22 fosticides . Widesprsticides . Candidasticides ,21.v channels (n = 33), Cav channels (n = 33), the lipid bilayer (n = 11), calcium-activated potassium (KCa) channels (n = 7), presynaptic nerve terminals (n = 2) and N-methyl-D-aspartate (NMDA) receptors (n = 1) [Spiders are among the most successful terrestrial predators. One reason for their success is the production of highly toxic venom from their venom glands that they employ to subdue prey and deter predators . Of the  (n = 1) . These t (n = 1) . Assumin (n = 1) .Spiders utilize their venoms to paralyze and/or kill prey or predators as rapidly as possible. Therefore their venoms are particularly rich in neurotoxins targeting the insect nervous system that rapidly modify ion conductance (ion channel toxins) and, to a lesser extent, affect neurotransmitter exocytosis (presynaptic toxins). Many of these spider peptide toxins are selectively insecticidal. In particular, insect-selective toxins have been patented for their possible use as bioinsecticidal agents for the control of phytophagous pests or insect vectors. Spider-venom peptides are a rich source of potential bioinsecticides that can combine the desirable attributes of high potency, novel target activity, structural stability and phyletic selectivity . MoreoveLycosa carolinensis) venom demonstrates both antimicrobial and insect neuroactive properties [Lycosa indicate some variation in peptide sequence is possible without losing biological activity [The selective spider venom peptide, lycotoxin-1 , from wolf spider  was designed to be cleaved by trypsin in the insect midgut to release the active 25-amino acid lycotoxin-1 peptide variant in the cuticle-lined gut region and allows for penetration into the insect. This variant differed from wild-type lycotoxin-1 in the two amino acids at the carboxy terminus, with proline and tryptophan residues replacing the lysine and leucine residues at positions 24 and 25, respectively, in the wild-type lycotoxin-1.Initial assays with lycotoxin 1 indicated it was inactive when fed to fall armyworms, a major insect pest of corn and other crops in the United States. Presumably this inactivity was the result of its inability to penetrate to the target site because of the oral delivery route. Normally spiders inject the peptides into their prey with their fangs, thereby bypassing the physical barrier of the insect cuticle. To investigate whether changes in the primary structure of lycotoxin-1 peptide might improve its efficacy as an oral or topical bioinsecticide, a gene library expressing recombinant mutant lycotoxin-1 peptides was produced using an amino acid scanning mutagenesis strategy  and screThe lycotoxin-1 variant that was highly lethal to the fall armyworms in this initial evaluation was selected for further optimization by amino acid scanning mutagenesis . The lycA helical-wheel projection of the amphipathic peptide illustrates the lycotoxin-1 mutations relative to the hydrophobic and hydrophilic sections of the three-dimensional structure , which codes for a 37-amino-acid calcium channel antagonist, was synthetically produced with codon optimization for plants and constitutively expressed in all tobacco tissues [Helicoverpa armigera, and cotton leafworm, Spodoptera littoralis, within two days. The same toxin from Hadronyche versuta was also effective against cotton bollworm, Helicoverpa armigera, caterpillars when expressed only in the phloem of transgenic tobacco [Macrothele gigas, predicted to be a 38-amino-acid mature peptide, with insecticidal properties but an unknown mode of action, was expressed in transgenic tobacco that conferred resistance to fall armyworm caterpillars [A small number of transgenic plants expressing spider toxins have been created in the last few years. The w-ACTX-Hv1a toxin gene from the Australian funnel web spider ( tissues . These t tobacco . A spiderpillars . Two of rpillars ,35, indiPest control is just one of the many potential uses of CPPs and CPP-like molecules. Recombinant technology and high-throughput platforms enable the rapid screening and selection of the most effective agent against a target pest. The possibility of encoding transgenes in a plant that serve a protective function against plant pests is a promising start in developing viable alternative biostrategies for plant pest control. For commercial success, it remains to be demonstrated that the potential agent is safe for non-target organisms, that it does not induce resistance, and that it can be produced inexpensively and in adequate quantities. However, with millions of possible bioactive peptides in the venom of spiders, and the application of molecular techniques to optimize efficacy and selectivity, an almost inexhaustible source of candidates is available.


The glutaraldehyde (GTA) cross-linked samples (GTA-chi group) were used for comparison. The 7-mm-diameter membrane implants made from either non-cross-linked chitosan or chemically modified materials with a cross-linking degree of around 80% were inserted in the ocular anterior chamber for 24 weeks and characterized by slit-lamp and specular microscopic examinations, intraocular pressure measurements, and corneal thickness measurements. The interleukin-6 expressions at mRNA level were also detected by quantitative real-time reverse transcription polymerase chain reaction. Results of clinical observations showed that the overall ocular scores in the GTA-chi groups were relatively high. In contrast, the rabbits bearing GP-chi implants in the anterior chamber of the eye exhibited no signs of ocular inflammation. As compared to the non-cross-linked counterparts, the GP-chi samples improved the preservation of corneal endothelial cell density and possessed better anti-inflammatory activities, indicating the benefit action of the GP cross-linker. In summary, the intracameral tissue response to the chemically modified chitosan materials strongly depends on the selection of cross-linking agents.Chitosan is a naturally occurring cationic polysaccharide and has attracted much attention in the past decade as an important ophthalmic biomaterial. We recently demonstrated that the genipin (GP) cross-linked chitosan is compatible with human retinal pigment epithelial cells. The present work aims to further investigate the  Over the past few years, the anterior chamber of a rabbit eye model was used in our laboratory to test the ocular biocompatibility of various kinds of materials such as amniotic membrane [co-poly(acrylic acid) [g-poly(N-isopropylacrylamide) [It is well known that biocompatibility is an important factor in determining the success of new medical implants and devices in the physiological environment. The word “biocompatibility” refers to the ability of a material to perform with an appropriate host response in a specific situation . Althougmembrane , hyaluromembrane , gelatinmembrane , poly2-ic acid) , and gelylamide) . A metho-ic acid d-glucosamine units and has wide biomedical applications, such as tissue engineering/regenerative medicine [Gardenia jasminoides Ellis [et al. showed that cross-linking of chitosan membrane using GP reduced its tensile strain, swelling ratio, and enzymatic degradability [et al. reported that the GP cross-linked chitosan/gelatin blends with optimal composition were able to support neuroblastoma cell adhesion and proliferation [et al. also demonstrated that the controlled release of bovine serum albumin from GP cross-linked chitosan microspheres could be useful for the tailoring of a protein drug delivery system [Chitosan, a naturally occurring cationic polysaccharide, is obtained by the deacetylation of chitin. It is primarily composed of repeating medicine ,8 and comedicine ,10. Crosmedicine . Genipines Ellis . Becausedability . A studyferation . Karnchay system .i.e., inchin-ko-to) in preventing posterior capsule opacification, and concluded that its main bioactive component, GP, may suppress α-TN4 lens cell fibrogenic behaviors [et al. showed that a GP cross-linking technique is useful to treat corneal ectasia and diseases involving corneal melting, due to a significant increase in biomechanical strength of collagenous tissue [Although the ophthalmic application of GP treated chitosan is rarely found in the literature , the potehaviors . Meanwhiehaviors . Recentls tissue . These fin vitro responses of retinal pigment epithelial cells to chemically modified chitosan materials [in vivo biocompatibility of GP treated chitosan (GP-chi group) by adopting an appropriate animal model. The glutaraldehyde (GTA) cross-linked samples (GTA-chi group) were used for comparison on the effect of cross-linker type. The 7-mm-diameter membrane implants made from either non-cross-linked chitosan or counterparts with cross-linking degree of around 80% were inserted in the ocular anterior chamber. During the follow-up period of 24 weeks, the intracameral tissue reaction was analyzed by slit-lamp and specular microscopic examinations, intraocular pressure measurements, and corneal thickness measurements. The inflammatory response was also monitored by interleukin-6 (IL-6) expressions. To the best of our knowledge, this is the first report to assess the ocular biocompatibility of GP cross-linked chitosan materials in the anterior chamber of the eye.In light of the promising results, it may be possible to develop GP cross-linked biomaterials for ocular therapeutics, tissue repair, and pharmacology. This situation has motivated us to extend our previous work in exploring the aterials . The aimin vivo degradation. The rabbit eyes showed very mild inflammation in the anterior chamber. After intraocular implantation of GTA-chi samples, severe ocular tissue responses, including aqueous flare, anterior chamber fibrin, corneal cloudiness, corneal neovascularization, iris neovascularization, and lens opacity were observed. By contrast, in the GP-chi groups, the implants appeared dark blue in color, mainly due to the formation of dark blue pigments via the reaction of GP with chitosan. These membranes remained stable in all rabbits at 24 weeks following surgery and demonstrated a slight reduction in size by 5%–10%. Of note, the eyes bearing the GP cross-linked materials showed no evidence of ocular inflammation.p > 0.05) with respect to aqueous flare, anterior chamber fibrin, corneal cloudiness, corneal neovascularization, iris neovascularization, and lens opacity measurements.The slit-lamp examination score for each group is shown in The morphology of corneal endothelial cells in rabbit eyes was observed by specular microscopy. As shown in 2. No significant difference was noted in the cell density of Ctrl groups (3145 ± 71 cells/mm2) compared with the values before surgery (p > 0.05). After exposure to the Chi implants for 24 weeks, the animals had a significantly lower cell density (2998 ± 63 cells/mm2). Because the biological responses were too severe to be observed, the endothelial cell counts of GTA-chi groups were not available from specular microscopic images. The cell density did not show a significant difference between the sham-operated and GP-chi groups .The IOP profile of the four groups is shown in p < 0.05). It was noted that the CCT was increased to a value larger than 1000 μm after implantation of GTA-chi samples in the ocular anterior chamber for 24 weeks.The CCT changes of the surgical corneas of the four groups are shown in Chitosan (Cat. No. 50494), derived from crab shell, is a commercial powder supplied by Fluka . According to information from the manufacturer, the chitosan samples used as raw materials had a degree of deacetylation of 95%–98% and a molecular weight of approximately 150 kDa. Genipin was purchased from Wako Pure Chemical Industries . Glutaraldehyde was obtained from Sigma-Aldrich . Phosphate-buffered saline  was purchased from Biochrom AG . TRIzol reagent was obtained from Gibco-BRL . Deionized water used was purified with a Milli-Q system . All the other chemicals were of reagent grade and used as received without further purification.v/v aqueous acetic acid (50 mL) with stirring until complete dissolution. To remove insoluble substances, the solution was passed through a filter paper . Then, 0.5 mL of chitosan solution was poured into a well of a 24-well plate  and air-dried for 2 days at 25 °C to obtain membranes (about 10 μm thick). The membrane samples were incubated in a 0.5 N NaOH solution for 1 h and washed extensively with deionized water until neutrality.The chitosan membranes (Chi group) were prepared by the solution casting method. In brief, chitosan (1 g) was added to 1% in vacuo for 24 h and sterilized in a graded series of ethanol solutions and thoroughly rinsed in sterilized PBS for use in the in vivo experiments.The chitosan materials were treated with GTA (GTA-chi group) or GP (GP-chi group) by respectively immersing the membrane samples in 5 mL of PBS containing 10 mM cross-linker. The cross-linking reaction was allowed to proceed at 25 °C for different time periods. To eliminate the residual GTA or GP, the cross-linked membranes were washed extensively in deionized water and cut out using a 7-mm-diameter corneal trephine device. Then, the samples were dried Cb) and after (Ca) cross-linking is proportional to the optical absorbance of the solution. The extent of cross-linking of the chitosan membranes was calculated as cross-linking index (%) = ((Cb − Ca)/Cb) × 100. Results were averaged over five independent runs. In this study, the GTA and GP treated implants with the same extent of cross-linking  were characterized by various in vivo assays.The amount of free amino groups of chitosan membranes was determined to evaluate their extent of cross-linking. The test sample was weighed and heated with a ninhydrin solution for 20 min. After the test solution was cooled to room temperature and diluted in 95% ethanol, the optical absorbance of the solution was recorded with a UV-visible spectrophotometer  at 570 nm using glycine at various known concentrations as standard ,22. The All animal procedures were approved by the Institutional Review Board and were performed in accordance with the ARVO  Statement for the Use of Animals in Ophthalmic and Vision Research. Twenty-four adult New Zealand white rabbits , weighing from 3.0 to 3.5 kg and 16–20 weeks of age, were used for this study. Animals were healthy and free of clinically observable ocular surface disease. A surgical operation was performed on a single eye of each animal, with no procedure being performed on the other eye. In the three test groups  of animals (six rabbits/group), the chitosan implants were inserted in the anterior chamber of the eye. The remaining six rabbits received no implant  and served as a control group (Ctrl).The rabbits were anesthetized intramuscularly with 2.5 mg/kg body weight of tiletamine hydrochloride/zolazepam hydrochloride mixture  and 1 mg/kg body weight of xylazine hydrochloride , and topically with two drops of 0.5% proparacaine hydrochloride ophthalmic solution . After disinfection and sterile draping of the operation site, the pupil was dilated with one drop of 1% atropine sulfate ophthalmic solution , and a lid speculum was put in place. Under the surgical microscope , the cornea was penetrated near the limbus by using a slit knife. Then, the corneal/limbal incision was enlarged to 7.5 mm with corneal scissors to allow the insertion of an implant in the anterior chamber. The incision site was finally closed with 10–0 nylon sutures.To determine the implant-tissue interaction in the anterior chamber, the rabbits were anesthetized under the same conditions as for surgery. Ophthalmic evaluations were performed before and 24 weeks after surgical insertion of material implants. The morphology of anterior segment of the eye including corneal and lens clarity, the degree of anterior chamber activity, iris, and implants was observed by slit-lamp biomicroscopy .The ocular grading method used for biomicroscopic examinations is shown in The corneal endothelial cell density in rabbit eyes was measured by specular microscopy . Each data point is an average of three independent observations.The intraocular pressure (IOP) was measured using a Schiotz tonometer , calibrated according to the manufacturer’s instructions. For each IOP determination, five readings were taken on each eye, and the mean was calculated.Central corneal thickness (CCT) was determined using an ultrasonic pachymeter  with a hand-held solid probe. During the measurements, the probe tip of the pachymeter was held perpendicular on the central cornea. An average of ten readings was taken.2 gas. The excised rabbit corneas were then processed for quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) analyses. Under a dissecting microscope , the Descemet’s membrane with the attached endothelium was aseptically stripped from the corneal stroma and washed three times with PBS  was performed using ImProm-II  and Oligo(dT)15 primers (Promega). The sequences of the primer pairs for each gene are listed in At the end of experiments, the animals were euthanized with COwith PBS . For in p < 0.05.Results were expressed as mean ± standard deviation. Comparative studies of means were performed using one-way analysis of variance (ANOVA). Significance was accepted with in vitro model based on ARPE-19 cell line culture [Biocompatibility is a prerequisite for the development of potential ophthalmic biomaterials. It has been recognized that biocompatibility is controlled mainly by the interface between foreign materials and host living cells/tissues . In our  culture . The resi.e., only corneal/limbal incision) does not cause signs of ocular inflammation and change in corneal endothelial cell morphology and density at 24 weeks post-operation. This is probably due to the fact that the initial responses to implantation of biomaterials are the acute and subacute phases of inflammation induced by surgical trauma [et al. have demonstrated that the chitosan films exhibit almost no degradation during 8 weeks of in vitro incubation in lysozyme solution [in vivo degradation. Mi et al. have reported that while the intramuscularly injected fresh chitosan microspheres retrieved at 12 weeks post-operation are already degraded into fragments, the degradation of the GP cross-linked chitosan counterparts is not significant after 20 weeks of implantation [Ophthalmic examinations including slit-lamp microscopy and specular microscopy were performed in all eyes to evaluate the status of the anterior segment. In the Ctrl groups, the sham operation (solution . The resantation . Our preantation . One posin vitro ocular biocompatibility of non-cross-linked chitosan membranes is demonstrated by the absence of any signs of toxicity or inflammation reaction [in vitro cell culture models have fewer inherent variations than the corresponding in vivo animal model, the in vitro toxicological studies are usually aimed at investigating the short-term effects of materials on acute toxicity. In our laboratory, an indirect contact methodology has also been used to evaluate cytotoxicity of biopolymers such as gelatin [in vitro experiments, the in vivo tests can more accurately determine the material biocompatibility, but they are usually more expensive and elaborate. During 24 weeks of follow-up evaluations, the chitosan implants were directly in contact with the tissues of the anterior segment of the eye. The continued residence of material samples in the intraocular cavity may elicit a much greater response.The reaction . However gelatin  and hyal gelatin  by a 2-det al. have demonstrated that GP exhibits inhibitory effect on nitric oxide production through the inhibition of nuclear factor-κB activation [et al. have also reported that the treatment of GP to lipopolysaccharide-stimulated microglia is effective at decreasing nitric oxide release, and suggested that the aglycon of geniposide can inhibit microglial activation in a mouse model of brain inflammation [et al. have shown that GP up-regulates heme oxygenase-1 via PI3-kinase-JNK1/2-Nrf2 signaling pathway to enhance the anti-inflammatory capacity in RAW264.7 macrophages [In the GTA-chi groups, the implants induce undesirable host reactions and some adverse biological effects, suggesting that the GTA cross-linking produces a strong influence on the interaction between chitosan materials and ocular tissues. Given that the GTA has very reactive properties and is easily interacted with cell surface, such a cross-linker can result in cytotoxicity and apoptosis . Our fintivation . Nam et ammation . More rerophages .in vivo. The effects of biomaterial implants on the endothelial cell morphology and count are also evaluated in relation to cross-linking agent type. The exposure of rabbit eyes to the GTA cross-linked chitosan implants causes severe tissue responses, whereas no adverse inflammatory reaction is noted after contact with GP treated chitosan samples. Although the endothelial cell count was not statistically different between the Ctrl and GP-chi groups, some irregularly shaped corneal endothelial cells and tiny dark areas were noted in The corneal endothelium is a thin cell monolayer that forms the posterior boundary of the cornea and maintains corneal clarity . Cellulain vivo presence of chitosan materials may increase aqueous humor outflow resistance in the trabecular meshwork, the rabbits bearing the GTA cross-linked chitosan implants reveal additional elevated IOP. These findings imply poor biocompatibility of GTA-chi samples, which corroborates biomicroscopic data. On the other hand, given that the corneal endothelial cells maintain corneal hydration and transparency by a pump-leak mechanism, CCT is considered as an indicator associated with corneal diseases [It is known that the IOP is an important measure of the balance between aqueous humor production and drainage. The disturbance of aqueous humor circulation is observed when the biomaterial implants are placed in the ocular anterior chamber . Our prediseases . The incNaturally occurring polymeric materials, including collagen , gelatinin vivo biocompatibility of GP-chi implants was shown at post-operative 24 weeks, the GTA-chi implants caused significant intraocular inflammation. The applications of GP cross-linked chitosan nanoparticles for drug delivery in the treatment of posterior segment diseases are currently being investigated.In conclusion, using the anterior chamber of a rabbit eye model, we found that the intracameral tissue response to the chemically modified chitosan materials strongly depended on the selection of cross-linker. While satisfactory


Anopheles gambiae, depends on availability of suitable surface water for oviposition. Short and long dry spells occur throughout the year in many parts of its range that limit its access to oviposition sites. Although not well understood, oviposition-site deprivation has been found to rapidly reduce egg batch size and hatch rate of several mosquito species. We conducted laboratory experiments to assess these effects of oviposition-site deprivation on An. gambiae and to evaluate the role of nutrition and sperm viability as mediators of these effects.The African malaria mosquito, Anopheles gambiae adults (1–2 d old) from the G3 laboratory colony were assigned to the following treatment groups: oviposition-deprived (fed once and then deprived of oviposition site for 7 or 14 d), multiple-fed control , and age matched blood-deprived control . Egg batch size and hatch rate were measured. In the second experiment two additional treatment groups were included: oviposition-deprived females that received either a second  blood meal or virgin males  4 days prior to receiving water for oviposition.An. gambiae was highly sensitive to oviposition-site deprivation. Egg batch size dropped sharply to 0–3.5 egg/female within 14 days, due to reduced oviposition rate rather than a reduced number of eggs/batch. Egg hatch rate also fell dramatically to 0-2% within 7 days. The frequency of brown eggs that fail to tan was elevated. A supplemental blood meal, but not ‘supplemental insemination,’ recovered the oviposition rate of females subjected to oviposition-site deprivation. Similarly, a supplemental blood meal, but not ‘supplemental insemination,’ partly recovered hatch rate, but this increase was marginally significant (P < 0.069).An. gambiae populations via reduced fecundity and fertility. However, females taking supplemental blood meals regain at least some reproductive success. If mosquitoes subjected to oviposition-site deprivation increase the frequency of blood feeding, malaria transmission may even increase during a short dry spell. The relevance of oviposition-site deprivation as a cue to alter the physiology of An. gambiae during the long dry season is not evident from these results because no reduction in hatch rate was evident in wild M-form An. gambiae collected in the dry season in the Sahel by previous studies.Even a short dry spell resulting in oviposition-site deprivation for several days may result in a dramatic decline of  Anopheles gambiae, the African malaria mosquito, depends on availability of suitable surface water for oviposition. However, during the dry season and dry spells during the wet season, larval sites may not be available for days, weeks, or even months in different environments[Like most mosquitoes, ronments-6. Underronments, which dronments-14.Aedes aegypti, Ae. albopictus, Culex quinquefasciatus, An. maculatus, and An. pharoensis[An. pharoensis) to 10 weeks , however, statistical analysis was not always applied to the data. Both An. pharoensis[An. maculatus[Only a few studies have evaluated the effects of oviposition-site deprivation on life-history traits. Reduced egg-batch size and hatch rate following oviposition-site deprivation was observed in haroensis,15-19. Imaculatus revealedAn. gambiae under laboratory conditions. We attempt to separate the effects of aging due to time and due to cumulative gonotrophic cycles from the oviposition-site deprivation itself and also evaluate the effect of sperm viability and the female nutritional state on the response to oviposition-site deprivation. Because this species experiences oviposition-site deprivation naturally, we hypothesized it would be less sensitive than other anopheline species previously examined. Our results suggest, however, that even short-term oviposition-site deprivation reduces egg batch size and hatch rate of An. gambiae and that nutrition is a key mediator of this effect.Here we evaluate the effects of oviposition-site deprivation on the egg batch size and hatch rate of An. gambiae was used throughout. Mosquitoes were reared under standard insectary conditions . Adults received fresh sugar solution daily. In each experiment, a total of 2,000 mosquitoes were divided, at 1–2 days old, into cages consisting of 100 females and 100 males to allow mating  were used to blood feed for twenty minutes; mosquitoes were provided with an additional twenty minutes if the feeding rate was <90%. Two-3-month old chickens were used to feed the mosquitoes in the cages, each one for a complete set of replicate cages . Unfed females were removed six hours after blood feeding.The G3 colony of On day seven, females to be assayed for oviposition  were separated into individual 50 mL conical tubes lined with absorbent paper for egg laying and secured with netting on top to allow access to sugar pads. Males were also removed from all cages on day seven. Water for oviposition (10 mL of dechlorinated tap water) was added individually to the tubes four days after blood feeding (no oviposition-site deprivation), 11 days after blood feeding (short-term: 7-day oviposition-site deprivation), or 18 days after blood feeding (long term: 14-day oviposition-site deprivation). Control groups included females that were blood-fed regularly  and those that were blood-fed only once (four days before water for oviposition was provided). Egg dishes were placed in the cages of the multiple-fed controls to allow the remaining females to lay eggs. Females that did not receive a blood meal on day 4 were considered blood-deprived (BD). These groups were fed once on day 11 or 18 and were provided with water for oviposition four days after feeding. Every treatment was replicated two or three times in each experiment  and hatch rate were determined (see below). Females that died before their phenotype could be determined were excluded from analysis. The tubes were checked twice a day for three days; if eggs were laid, the female was killed and preserved and the appearance and number of eggs was recorded. Eggs that had sunk into the water were recorded separately and excluded from the count because they appeared abnormal and did not hatch in previous studies,21. EggsThe second experiment was carried out as stated above, with the addition of two treatment groups (each replicated twice). One treatment consisted of oviposition-deprived females that were provided with a second  blood meal 4 days prior to receiving water for oviposition. The other treatment consisted of oviposition-deprived females, which received 100 4-day-old virgin males as supplemental mating. The males were kept in the cage for 3 days until the females were separated into oviposition tubes. A random collection of males from the same cohort and cages was placed with virgin females to assess their mating vigor. Virtually all virgin females were inseminated after 3 days . Individual females were separated and EBS and hatch rate were determined at the same time points as stated above.This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal procedures were approved by the National Institutes of Health Animal Care and Use Committee .st and 2nd gonotrophic cycle (1 contrast), and between the oviposition-deprivation group and the age-matched blood-deprivation group which received their first and only blood meal 3 days prior to the water for oviposition (1 contrast). The third contrast compared the EBS of the 1st and 2nd gonotrophic cycle of the non oviposition-deprived controls. Because all females laid no (zero) eggs in some treatment/replicate combinations, resulting in non-estimable hatch rates, the variance component associated with ‘replicate’ could not be estimated in such cases. To determine differences in the percentage of females that laid eggs from different groups, log-linear categorical models were implemented using PROC CATMOD, which allows testing the same contrasts.Statistical analyses were performed using SAS 9.2 . The effects of the treatments on EBS and hatch rate were evaluated using mixed model ANOVA (implemented using PROC MIXED) because both fixed-effect variables  and random variables  were included. If significant differences were indicated between treatments overall, contrasts were used to specifically evaluate the effect of oviposition-site deprivation compared with a subset of controls. Thus, to assess the effect of 7-day oviposition-site deprivation on EBS, contrasts were made between that group and the non oviposition-deprived controls which were in their 1An. gambiae was determined by comparison of EBS and egg-hatching rate of females subjected to short (7 days) and long (14 days) oviposition-site deprivation with corresponding values of non-deprived females of the same gonotrophic cycle as well as those of the same  age but in the next gonotrophic cycle. Additionally, oviposition-deprived females were compared to females of the same chronological age and same gonotrophic cycle, which received their first blood meal 3 days before they were offered water for oviposition; the last group of females was deprived of a blood meal (blood deprivation). Unless otherwise stated, data obtained in the two experiments were analyzed together, treating experiments as random blocks (see Methods).The effect of oviposition-site deprivation on the reproductive output of 1,147 = 150.8), which probably explains their smaller overall EBS (across groups) and reduced the statistical power due to smaller differences between groups. Even short-term oviposition-site deprivation (7 day) reduced mean EBS approximately 2-fold compared with that of the first and second gonotrophic cycles  reduced mean EBS several fold, to near zero, compared with that of the first and second gonotrophic cycles  was significantly reduced compared with non-deprived females, but the difference in mean EBS between the oviposition-site deprived and non-deprived females in those that laid eggs was minimal  oviposition-site deprivation laid no eggs (see above), precluding estimating hatch rate for these groups. Thus, hatch rate was analyzed considering short-term (7-day) oviposition-site deprivation only. Remarkably, short-term oviposition-site deprivation reduced hatch rate to 0-2% from 60-75% in both experiments , but not after they received “supplemental insemination” Figure. The hatAn. gambiae to short-term oviposition-site deprivation suggests that even short dry spells (measured in days) during the wet season can reduce reproductive success, especially because of lower fertility (hatch rate) augmented by lower fecundity (egg batch size) due to a lower rate of oviposition. Similar sensitivity was reported for other culicids including An. pharoensis and An. maculatus[An. gambiae. It is possible that a colony adapted to laboratory conditions has increased its sensitivity to oviposition-site deprivation because such deprivation is too rare in an insectary and it may also be related to other factors such as different blood source (chicken instead of human in our colony). However, the agreement across anopheline species (above), especially with the arid-adapted An. pharoensis, which was raised in the laboratory only three years prior to the study and was provided with human blood, suggests that the sensitivity to oviposition-site deprivation reflects the natural response of anophelines to such pressure.The sensitivity of maculatus,15-18. HAn. gambiae females are required to clarify that hypothesis.However, oviposition-site deprivation was linked, as in most other studies listed above, with no (or limited) access to blood meals. The capacity to regain reproductive success by supplemental blood feeding suggests that, under natural conditions, mosquitoes may avoid the effects of oviposition-site deprivation by increasing their blood-feeding frequency. This hypothesis is consistent with the observations of gravid females that take additional blood meals,23,24. TAn. gambiae during the long dry season is not evident from these results. Similar to this study, Yaro et al. (2012)[Culex pipiens quinquefasciatus and West Nile Virus[The importance of oviposition-site deprivation as a cue to alter the physiology of . (2012) reported. (2012) and a hi. (2012). Althougle Virus.The authors declare that they have no competing interests.KLD participated in the study design, carried out the experiments, and helped draft the manuscript. DLH participated in the study design and helped carry out various aspects of the experiments as well as draft the manuscript. TL conceived of the study, participated in its design and coordination, conducted the statistical analysis, and drafted the manuscript. All authors read and approved the final manuscript.Figure S1. Variation in body size, measured by wing length (WL), within and among treatment groups in the two experiments. The dotted horizontal line denotes the average WL across all groups of each experiment. Sample sizes smaller than 10 are denoted by numbers under corresponding box-whisker plots.Click here for fileFigure S2. Partitioning the effect of oviposition-site deprivation on overall egg batch size , the number of eggs per batch for females that laid eggs (middle panel), and oviposition rate (lower panel). EBS of the treatment groups was averaged across experiments (see FigureClick here for file


The volumes of the manual and semi-automated segmentations did not statistically differ , were highly correlated  and showed a very strong spatial similarity . In conclusion, our semi-automated method to measure the load of WMH is highly reliable and could represent a good tool that could be easily implemented in routinely neuroimaging analyses to map clinical consequences of WMH.White matter hyperintensities (WMH) are brain areas of increased signal on T2-weighted or fluid-attenuated inverse recovery magnetic resonance imaging (MRI) scans. In this study we present a new semi-automated method to measure WMH load that is based on the segmentation of the intensity histogram of fluid-attenuated inversion recovery images. Thirty patients with mild cognitive impairment with variable WMH load were enrolled. The semi-automated WMH segmentation included removal of non-brain tissue, spatial normalization, removal of cerebellum and brain stem, spatial filtering, thresholding to segment probable WMH, manual editing for correction of false positives and negatives, generation of WMH map, and volumetric estimation of the WMH load. Accuracy was quantitatively evaluated by comparing semi-automated and manual WMH segmentations performed by two independent raters. Differences between the two procedures were assessed using Student’s  White matter hyperintensities (WMH) are brain areas of increased signal intensity, appearing on T2-weighted (T2-w) or fluid-attenuated inversion-recovery (FLAIR) magnetic resonance imaging (MRI) scans, that result from localized changes in tissue composition . The etiWMH are often observed in elderly participants , and theAlthough neuropathological, clinical, and cognitive significance of WMH is unclear  large epInterestingly, a systematic review and meta-analysis  providesA challenging issue in studying WMH is their quantification and localization, given their variability and scattered spatial distribution. Two analytic strategies have been used to evaluate WMH on MRI brain images: (1) semi-quantitative rating systems and (2) quantitative volumetric analyses. The semi-quantitative approach mainly consists in the computation of visual ratings and several scales, markedly different in morphological or anatomical definitions, have been proposed and used in literature . WMH visOn the other hand, a number of more recent researches have introduced quantitative methods of measuring WMH severity using computer-based techniques to obtain volumetric measures of WMH burden. These methods vary from manual outlining techniques to fully-automatic WMH detection .Manual outlining techniques are referred to as region-of-interest (ROI) methodologies, in which the tracer inspects the scan using visualization softwares and manually draws WMH areas. The volume of each region is then calculated from the section thickness and the number of voxels included in the traced area. The values of all sections are then added together to make a total WMH volume. Manual outlining procedures, although accurate, have several limitations such as labor intensiveness, time consume, subjectiveness, and error-proneness. In addition, manual detection suffers from intra-observer and inter-observer variability .In recent years, major progresses have been made on the development of semi- or fully-automated segmentations of WMH. These techniques are based on computer algorithms developed to replace human eye, allowing the collection of quantitative volumetric data on WMH . These tHowever, such methods have different accuracy, computational speed, and complexity. The majority of these approaches relies on multimodal data, thus being based on different MRI sequences, including T2-w, proton density (PD), T1-weighted (T1-w), inversion recovery (IR) and, more widely, FLAIR images. Innovative procedures to detect WMH have been developed based on Markov random field model , k-neareAll the quantitative methods here discussed have some limitations. Indeed, complex automatic or semi-automatic computer-based segmentation procedures require a large amount of technical resources that may not be available in clinical settings. Furthermore, multi-spectral approaches are not always accessible in clinical practice since the acquisition of all these images is cost-intensive and requires long processing time . ConversIn this study, we present a semi-automated method for WMH load measurement that overcomes the above presented limits. We applied the method on a group of MCI patients with various WMH load. The aims of our study are: (i) to develop a semi-automated algorithm for the detection, quantification, and localization of WMH using only FLAIR images and T1-w at 3T which is fully reproducible in different research contexts and clinical populations; and (ii) to evaluate the accuracy of the method by comparing it with manual segmentation.We recruited in our memory clinic in Rome, Italy, 30 patients with amnestic mild cognitive impairment  major medical illnesses and autoimmune-inflammatory disease; (2) co-morbidity of primary psychiatric or neurological disorders, and (3) any other significant mental or neurological disorder. Medical and psychiatric histories were obtained from each subject, and all patients underwent a series of standard clinical examinations, including physical, neurological and mental status examinations, neurocognitive tests, and brain MRI (described below). All patients underwent the first diagnostic assessment and none were taking acetylcholinesterase inhibitors or psychotropic drugs . The study was approved and undertaken in accordance with the guidance of our local Ethics Committee and written consent was obtained from all participants.All 30 participants underwent the same imaging protocol, which included 2D FLAIR and whole-brain high-resolution T1-w images, using a 3T Allegra MR imager  with a standard quadrature head coil. All planar sequences acquisitions were obtained in the plane of the AC–PC line. Particular care was taken to center the subjects in the head coil and to restrain their movements with cushions and adhesive medical tape.Two-dimensional FLAIR images were obtained in the axial plain . Whole-brain T1-w images were obtained in the sagittal plane using a modified driven equilibrium Fourier transform . All scans were visually inspected by a neuroradiologist (GL) with high expertise in clinical neuroimaging in order to exclude images with poor quality and motion artifacts.Figure 1.A representative axial slice of FLAIR images for each patient is shown in 1 and the VBM8 toolbox2 implemented in SPM 83 running in Matlab 2007b  and it consisted of three major steps:The semi-automated WMH segmentation algorithm was performed using FSL 4.1(1)Image pre-processing, including non-brain tissue removal, spatial normalization into MNI space, removal of cerebellum and brain stem, and spatial filtering;(2)Automated detection of WMH in FLAIR images and, if necessary, subsequent manual editing for removal of false positives;(3)Post-processing, including the generation of WMH map and volumetric estimation of the WMH load at the individual and sample levels.Image preprocessing included skull-stripping of the FLAIR images to restrict our analyses to brain tissue only reducing the risk of false positives and to improve the accuracy and efficiency of WMH segmentation.For each subject, the FLAIR image was co-registered to the T1-weighted image using the FLIRT tool, integrated within FSL software through full affine alignment with nearest-neighbor resampling (correlation ratio cost function). Then, VBM8 with standard options was used to segment T1-weighted images and obtain a whole binary brain mask by summing the segmented GM, WM, and CSF in native space. The mask was then multiplied by the co-registered FLAIR and T1-w images to remove non-brain tissue. The average time for this step was 15 min per subject.The T1-w skull-stripped images were then linearly registered to MNI152 T1 1 mm brain template, and the transformation matrix was applied to the skull-stripped FLAIR images. Average time to complete the task for each subject was 1 min.Since WMH are rare in cerebellum and brain stem , we creaStep 1 was performed using an in-house shell script which automatically concatenates all three substeps for all subjects.For each subject we used FSL fslstats utility to calculate the mean and SD of the intensity of all brain voxels. In our procedure, after several trials, we optimized the process by choosing as threshold value the intensity mean +1.5 SD. This threshold was computed and applied to the FLAIR image for each subject, thus generating a map (of likely WMH) excluding all voxels having an intensity below such threshold. The time to complete the task was 1 min per subject and was implemented in a “for” cycle within a shell script.Figure 2, middle row) by a trained neuropsychologist (FP) with a strong expertise in lesion analyses using the image processing software MRIcron4. In particular, wrong identifications of WMH were deleted through the eraser tool of MRIcron, while areas of WM lesions that were omitted by the algorithm were re-included. Correction of false positives was necessary for two patients while false negatives were present in nine patients  and mainly consisted of underestimation of periventricular WMH volume. Manual correction required an average time of 8 min per patient.Each generated WMH map was visually inspected and compared to the original FLAIR image in order to identify false classifications (positive or negative). These were then manually corrected (as shown in 3) were calculated automatically using FSL fslstats utility, again trough an automatic shell script developed in-house.The final output provided by the system is a binary image in which a voxel is valued 1 if it is considered a WMH, 0 otherwise. Using these binary masks, for each subject, WMH volumes  were calculated automatically using FSL fslstats utility. The mean time to complete the task for each subject was 2 h and 32 min.The manual segmentation of WMH on FLAIR images was performed by an expert neuroradiologist (Giacomo Luccichenti) and a trained clinician (Claudia Cacciari), expert in lesion segmentation, who were not aware of the results of the semi-automated procedure. Manual segmentation was delineated on the standard registered FLAIR images using MRIcron software by tracing the lesion outline with a mouse-controlled interface. This process resulted in the definition of binary images, considered as t-test while a linear regression model was computed to assess the relationship between the two datasets.Statistical analyses were performed with Statview software. The inter-rater reliability was calculated using the Spearman correlation coefficient. The differences between volumetric data derived from semi-automated and manual segmentations were evaluated using Student’s Furthermore, we assessed the similarities between the two datasets in terms of both volumetric and spatial agreement by computing the Dice similarity coefficient DSC; , which iwhere Seg is the automatically segmented image and Ref is the manually segmented image. DSC reflects differences in locations more strongly than differences in size . Indeed,3). The mean (±SD) volume of WMH from the two manual raters was 16.74 (±13.89) cm3 and 19.5 (±16.29) cm3 per subject (see Table 1).The semi-automated procedure of WMH quantification was successfully completed in all cases. Hyperintense signals on FLAIR images were present in all subjects, but their extent and distribution varied considerably . Further, no statistical differences were found in the comparison between semi-automated and manual rater 1 WMH segmentation volumes  as well as between semi-automated and manual rater 2 WMH segmentation volumes .The two raters performed similarly in manual lesion segmentation  = 155.540, p < 0.0001 for the manual rater 1 and r = 0.967, r2 = 0.935, F = 402.709, p < 0.0001 for the manual rater 2.Furthermore, as shown in Table 2).Finally, the two WMH segmentation procedures showed a very strong spatial similarity, with high DSC .In the present study, we showed our semi-automated procedure for the detection, localization, and quantification of WMH on FLAIR images applicable to a wide range of patients with various diseases. This procedure is based on FLAIR and T1-w images , since no statistical differences between the two outputs were found and a very high similarity emerged, both in terms of volumetric load and spatial location. This is an outstanding outcome, since the semi-automated procedure requires a time consumption which is approximately six times lower than the manual approach.Other automated procedures developed to classify and quantify WMH have used a variety of classification approaches, including Markov random field model , k-neareFirst, we incorporated specific steps without any human intervention including the removal of non-brain tissue and of areas where WMH are unlikely  in order to improve the accuracy of the method and overcome the limitations of subjectivity of the raters, thus ensuring test–retest reliability. Furthermore, these steps, which are not included in the majority of other methodologies, reduce the probability to include false positives and decrease the time spent to remove them, thus resulting in faster processing and more accurate maps of WMH.Second, the transformation of FLAIR images from native to standard space (MNI), allows later statistical group comparisons and/or voxel-based statistics since the WMH images are in a common coordinates system. This feature enables a quantitative estimation and localization of WMH that can be further processed using statistical voxel-based image analysis softwares, which may be of importance for the study of the consequences of WM changes on cognition and behavior . Indeed,Moreover, our automated approach offers the possibility of a fully streamlined methodology with little user intervention and utilizes freely available software , which can be easily implemented on most desktop computers of standard power and are of routine use in most laboratories equipped to carry out neuroimaging research. This makes our approach reasonably accessible. Relatively little image manipulation is required and the quantification approach is procedurally straightforward. Additionally, little knowledge in computer programming allows to automate the steps and greatly reduce the processing time using in-house shell scripts which automatically concatenate all steps for all subjects.A possible limitation of the presented methodology lies in the low number of brain slices used as inputs. In fact, the analyzed FLAIR images were acquired with a slice thickness of 5 mm which may be a poor resolution for accurate volumetric quantification and might induce interpolation errors in the registration processes. The MRI scanner used in the present study is relatively old and does not allow fast high-resolution 3D FLAIR imaging. A higher image resolution could improve the WMH quantification, and the registration accuracy, which would lead to more precise WMH localization; however, it would lead to an increase of acquisition time thus decreasing clinical practicability. However, modern MRI systems allow fast 3D FLAIR acquisition which would surely improve image resolution and, in turn, WMH segmentation.In summary, we presented an automated detection and segmentation approach for WMH using T1-w and FLAIR images. By evaluating our method, we demonstrated that this segmentation approach is robust and reliable and we suggest that it has potential use in clinical studies.Future works should consider the presented method to map cognitive consequences of WMH and their progression in time through large (and possibly multicenter) cross-sectional and longitudinal studies.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.Mariangela Iorio designed the study, analyzed imaging data, contributed to the writing of the manuscript; Gianfranco Spalletta designed the study, analyzed data, and contributed to the writing of the manuscript; Chiara Chiapponi analyzed imaging data and contributed to the writing of the manuscript; Giacomo Luccichenti analyzed imaging data and contributed to the writing of the manuscript; Claudia Cacciari analyzed imaging data; Maria Donata Orfei wrote the draft of the manuscript; Carlo Caltagirone contributed to the writing of the manuscript; Fabrizio Piras designed the study, analyzed imaging data, contributed to the writing of the manuscript.


LTs are involved in inflammatory and allergic diseases like asthma, ulcerative colitis, rhinitis and also in cancer. Consequently 5-LOX has become target for the development of therapeutic molecules for treatment of various inflammatory disorders. Zileuton is one such inhibitor of 5-LOX approved for the treatment of asthma.Lipoxygenases (LOXs) are non-heme iron containing dioxygenases involved in the oxygenation of polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA). Depending on the position of insertion of oxygen, LOXs are classified into 5-, 8-, 9-, 12- and 15-LOX. Among these, 5-LOX is the most predominant isoform associated with the formation of 5-hydroperoxyeicosatetraenoic acid (5-HpETE), the precursor of non-peptido  strategies have been applied successfully in drug development processes. A comprehensive review on structure based drug design strategies in the development of novel 5-LOX inhibitors is presented in this article. Since the crystal structure of 5-LOX has been recently solved, efforts to develop 5-LOX inhibitors have mostly relied on ligand based rational approaches. The present review provides a comprehensive survey on these strategies in the development of 5-LOX inhibitors. AA can be metabolized by three pathways: cyclooxygenase (COX), lipoxygenase (LOX) and epoxygenase (EPOX) as depicted in Fig. (1).Arachidonic acid (AA) is normally found esterified to cell membrane glycerophospholipids. Activation of phospholipase A2), which is subsequently reduced by the peroxidase to the hydroxy endoperoxide, PGH2  hydroxamic acids. They observed a good correlation between the octonal-water partition coefficient of the group attached to the carbonyl of the hydroxamate and the potency for 5-LOX inhibition. Importantly, they revealed that hydrophobic groups close to the hydroxamic acid functionality do not contribute to increased inhibition and the hydrophobicity of fragments beyond approximately 12 Å from the hydroxamate do not influence potency. They also demonstrated that inhibitory activity was enhanced in cases where there was an alkyl group on the hydroxamate nitrogen, electron-withdrawing substituents and when the hydroxamate was conjugated to an aromatic system. These results provided a simple and a very reasonable description of the hydroxamic acid binding site in 5-LOX.In an earlier study in 1990, Summers et al.  performepharmacophore to refer to the molecular framework that carries (phoros) the features that are essential for the biological activity of a drug (pharmacon) phenazine showed an IC50 of 0.96 µM. These results encourage the use of pseudoreceptor modeling for virtual screening studies.Pseudoreceptor modeling has been successfully applied by Hofmann and his colleagues for the identification of novel inhibitors of 5-LOX . A serieet al. [As pointed out by Rödl et al.  the crysThe accuracy of the current CADD approaches for 5-LOX can be improved enormously by the study of protein flexibility, induced-fit adaptations, the role of water insolvation, desolvation and ligand binding. One of the major limitations in drug design process is the inability of the existing docking programs to accurately estimate binding affinities. Combination of docking protocol and scoring function to be employed requires validation before their application in a drug discovery project. In virtual screening processes, it is noticeable that some of the high scoring ligands miss interactions that are known to be important for the target receptor . ImproviThe most important challenge for pharmacophore based virtual screening is the percentage of virtual hits that are really bioactive. Usually the screening results bear a higher false positive and false negative rate. Many factors can contribute to this problem, including the quality and composition of the pharmacophore model and the available information of 5-LOX structure. The increase in number of potential inhibitors of 5-LOX, with activities derived from a uniform assay method would surely enhance the quality of these methods.in vitro studies showed toxic side effects in animal studies or human clinical trials. This may be due to non-specific binding to other LOX isoforms, 12-LOX and 15-LOX. LOXs have a conserved structural pattern, mainly at the catalytic site hence non-specific binding will remain a major issue. CADD approaches should be applied to understand the molecular details of the binding sites of LOX isoforms and salient features/patterns should be illustrated to further exploit in specific inhibitor design. With the availability of crystal structures of 5-LOX [From the case studies discussed in the review it is evident that the main focus has been on the development or identification of potential 5-LOX inhibitors. There has been limited attention on the development of isozyme specific 5-LOX inhibitors. As discussed, most of the potent inhibitors of 5-LOX identified from of 5-LOX , 12-LOX of 5-LOX , methods5-LOX is the key enzyme involved in the biosynthesis of leukotrienes, the mediators of allergy, asthma, GERD, Crohn's disease and other inflammatory disorders. 5-LOX is also associated with various cancers. As 5-LOX is implicated in many diseases, there is growing emphasis by many pharmaceutical companies and academic research groups on the development of effective 5-LOX inhibitors. Structure based and ligand based drug design approaches are being employed in 5-LOX drug development strategies. Lack of crystal structure information of 5-LOX, however, has been an obstacle for the application of structure based drug design strategies. Homology models of lipoxygenase enzymes have been used in several previous studies but there are fewer reports on their usage for inhibitor discovery. Specifically, homology models were sometimes used effectively to understand SAR of novel class of inhibitors by docking methods. There are few reports where homology model of 5-LOX has been used further in designing novel inhibitors. These studies resulted in the design of novel inhibitors with μM range activity. The recent elucidation of the crystal structure of 5-LOX should advance the 5-LOX inhibitor design in the coming years. It can be a working tool for more precise predictions of function and binding affinities of inhibitors. Ligand based drug design strategies have been widely employed to quantitatively explore common chemical characteristics among a considerable number of known 5-LOX inhibitors with great diversity. Results of pharmacophore modeling studies clearly suggest the importance of hydrogen bond acceptor, hydrophobic and aromatic interactions for 5-LOX inhibitory activity. Successful application of these pharmacophore models as a query for searching chemical databases and identification of new chemical entities supports their possible use in the assessment of binding affinities. The application of pseudoreceptor models and scaffold hopping in computer aided 5-LOX inhibitor design is also well demonstrated. Scaffold hopping will aid in finding new classes of compounds against 5-LOX and should be a main focus. CADD approaches have been so far applied in the development of specific 5-LOX inhibitors, however, in future more emphasis should be on the design of isozyme specific inhibitors which may have fewer or negligible side effects.