Evaluation of the basophil activation test and skin prick testing for the diagnosis of sesame food allergy.

BACKGROUND: The prevalence of sesame food allergy (SFA) has increased over recent years, with the potential of anaphylactic reactions upon exposure. Oral food challenge (OFC) remains the diagnostic standard, yet its implementation may be risky. Commercial skin prick tests (SPT) have a low sensitivity. Investigation of alternate diagnostic methods is warranted. OBJECTIVE: To evaluate the utility of SPT and the basophil activation test (BAT) for SFA diagnosis. METHODS: Eighty-two patients with suspected SFA completed an open OFC to sesame or reported a recent confirmed reaction. Patients were administered skin prick tests (SPT) with commercial sesame seed extract (CSSE) and a high protein concentration sesame extract (HPSE) (100 mg/mL protein). Whole blood from 80 patients was stimulated with sesame seed extract (40-10 000 ng/mL protein) for BAT), assessing CD63 and CD203c as activation markers. RESULTS: Sixty patients (73%) had IgE-mediated reactions to sesame, and 22 (27%) did not react. Receiver operating characteristic (ROC) curve analysis demonstrated an area under the curve (AUC) of 0.87 for HPSE-SPT and 0.66 for CSSE-SPT. At 1000 ng/mL of sesame protein, induction of CD63 and CD203c was weakly but significantly associated with OFC eliciting dose by rank (Spearman's rho = -.42 (P < .01) and -.35 (P < .05) for CD63 and CD203c, respectively). By ROC analysis, the AUC was 0.86 for CD63 and was 0.81 for CD203c sesame-induced basophil expression. Using HPSE-SPT as a first test to definitively diagnose (n = 24) or rule-out (n = 5) SFA and BAT as a second test to diagnose the remainder results in the correct classification of 73 of 80 (91%) patients, leaving one false negative and 4 false positive patients. Two BAT non-responders remain unclassified by this algorithm. CONCLUSIONS & CLINICAL RELEVANCE: While prospective cohort validation is necessary, joint utilization of BAT and SPT with HPSE extract may obviate the need for OFC in most SFA patients.

A Sperm Spawn-Inducing Pheromone in the Silver Lip Pearl Oyster (Pinctada maxima).

Pheromones are considered to play an important role in broadcast spawning in aquatic animals, facilitating synchronous release of gametes. In oysters, the sperm has been implicated as a carrier for the spawn-inducing pheromone (SIP). In hatchery conditions, male pearl oysters (Pinctata maxima) can be stimulated to spawn through a variety of approaches (e.g. rapid temperature change), while females can only be induced to spawn through exposure to conspecific sperm, thus limiting development of targeted pairing, required for genetic research and management. The capacity for commercial production and improvement of genetic lines of pearl oysters could be greatly improved with access to a SIP. In this study, we prepared and sequenced crude and semi-purified P. maxima sperm extracts that were used in bioassays to localise the female SIP. We report that the P. maxima SIP is proteinaceous and extrinsically associated with the sperm membrane. Bioactivity from pooled RP-HPLC fractions, but not individual fractions, suggests that the SIP is multi-component. We conclude that crude sperm preparations, as described in this study, can be used as a sperm-free inducer of female P. maxima spawning, which enables for a more efficient approach to genetic breeding.

NUT Co Reactivity - ACquiring Knowledge for Elimination Recommendations (NUT CRACKER) study.

BACKGROUND: Ambiguities exist regarding the diagnosis of tree-nut allergy, necessitating either their elimination or the performance of oral food challenges (OFCs). OBJECTIVE: To examine the coincidences of allergies among tree-nuts and improve diagnostic testing to minimize the need for OFC. METHODS: Eighty-three patients prospectively evaluated for walnut, pecan, cashew, pistachio, hazelnut, and almond allergy. A history of previous reactions was obtained, and standardized skin prick tests (SPTs) using finely ground tree-nut solution and basophil activation tests (BAT) were performed. Patients underwent OFC for each tree-nut they eliminated and to which a reaction in the previous 2 years was not documented. RESULTS: While most patients were sensitized to 5-6 tree-nuts, over 50% were allergic to only 1-2 tree-nuts. The highest rate of allergy in sensitized patients was observed for walnut (74.6%) and cashew (65.6%). The rate of co-allergy for most tree-nuts was <30%. Two-thirds of walnut- and cashew-allergic patients were also allergic to pecan and pistachio, respectively, while all pecan- and pistachio-allergic patients were allergic to walnut and cashew, respectively. Receiver-operating characteristic analysis for SPT and BAT was tree-nut dependent and yielded area under the curve (AUC) values ranging from 0.75 to 0.94. Knowledge of coincident allergies in these pairs along with the combination of SPT and BAT correctly distinguished allergic from tolerant patients for walnut (87%), pecan (66%), cashew (71%), and pistachio (79%). CONCLUSION: The data presented here should assist in differentiating between allergic and tolerant patients, decrease the need for OFC, and allow for appropriate elimination recommendations.

Changes in patient quality of life during oral immunotherapy for food allergy.

BACKGROUND: Quality of life (QOL) is impaired in patients with food allergy and improves following oral immunotherapy (OIT). However, the treatment itself is prolonged and demanding. We examined changes in patient QOL during OIT for food allergy. METHODS: The FAQLQ-PF was administered to children aged 4-12 years undergoing OIT for milk, peanut, or egg allergy, at the beginning and after 4 months of treatment. Patients were categorized as improved, unchanged, or diminished FAQLQ-PF (>0.5 point decrease, a change of ≤0.5 points, or >0.5 increase, respectively) and compared. Food-allergic patients not undergoing OIT served as controls. RESULTS: The Food Anxiety, Social and Dietary Limitation, and total FAQLQ-PF scores improved significantly during the study period (P=.001, P=.018, and P=.01, respectively) in treated but not in control patients, while the Emotional Impact did not. The change in the FAQLQ-PF was independent of the maximal tolerated dose at baseline or following four months of treatment, the pace of dose increase, or the number or severity of reactions experienced. The total FAQLQ-PF score was inversely associated with the score at baseline on multivariate analysis (regression coefficient=-0.56, P<.001). That was driven primarily by improvement in QOL scores in patients with high score (worse QOL) at baseline. Some patients with low FAQLQ-PF score (better QOL) at baseline deteriorated. CONCLUSIONS: QOL of patients with food allergy improves in some but deteriorates in others during OIT. Patients with impaired QOL at baseline improve significantly despite the treatment burden. Some patients with better QOL at baseline might deteriorate during OIT.

Clinical and laboratory 2-year outcome of oral immunotherapy in patients with cow's milk allergy.

Studies examining the long-term effect of oral immunotherapy in food-allergic patients are limited. We investigated cow's milk-allergic patients, >6 months after the completion of oral immunotherapy (n = 197). Questionnaires, skin prick tests, and basophil activation assays were performed. Of the 195 patients contacted, 180 (92.3%) were consuming milk protein regularly. Half experienced adverse reactions, mostly mild. Thirteen patients (6.7%) required injectable epinephrine. Higher reaction rate after immunotherapy was associated with more anaphylactic episodes before treatment and a lower starting dose (OR = 2.1, P = 0.035 and OR = 2.3, P = 0.035, respectively). Reaction rate in patients who were 6-15 months, 15-30 months, or >30 months post-treatment decreased from 0.28/month to 0.21/month to 0.15/month, respectively (P < 0.01). Milk-induced %CD63 and %CD203c expression was significantly lower in patients >24 months vs in patients <24 months post-treatment (P = 0.038 and P = 0.047, respectively). In conclusion, many patients experience mild adverse reactions after completing oral immunotherapy and some require injectable epinephrine. Progressive desensitization, both clinically and in basophil reactivity, occurs over time.

Primordial germ cell migration in the yellowtail kingfish (Seriola lalandi) and identification of stromal cell-derived factor 1.

Primordial germ cells (PGCs) are progenitors of the germ cell lineage, giving rise to either spermatogonia or oogonia after the completion of gonadal differentiation. Currently, there is little information on the mechanism of PGCs migration leading to the formation of the primordial gonad in perciform fish. Yellowtail kingfish (Seriola lalandi) (YTK) (order Perciforms) inhabit tropical and temperate waters in the southern hemisphere. Fundamental details into the molecular basis of larval development in this species can be easily studied in Australia, as they are commercially cultured and readily available. In this study, histological analysis of YTK larvae revealed critical time points for the migration of PGCs to the genital ridge, resulting in the subsequent development of the primordial gonad. In YTK larvae at 3, 5, 7 and 10 days post hatch (DPH), PGCs were not yet enclosed by somatic cells, indicating the primordial gonad had not yet started to form. While at 15, 18 and 20 DPH PGCs had already settled at the genital ridge and started to become enclosed by somatic cells indicating the primordial gonad had started to develop. A higher number of PGCs were observed in the larvae at 15 and 18 DPH indicating PGCs proliferation, which corresponds with them becoming enclosed by the somatic cells. Directional migration of PGCs toward the genital ridge is a critical event in the subsequent development of a gonad. In zebrafish, mouse and chicken, stromal-cell derived factor (SDF1) signalling is one of the key molecules for PGC migration. We subsequently isolated from YTK the SDF1 (Slal-SDF1) gene, which encodes for a 98-residue precursor protein with a signal peptide at the N-terminus. There is spatial conservation between fish species of four cysteine residues at positions C9, C11, C34 and C49, expected to form disulphide bonds and stabilize the SDF structure. In YTK, Slal-SDF1 gene expression analyses shows that this gene is expressed in larvae from 1 to 22 DPH and demonstrates distinct spatial localisation in the larvae at 7 DPH. These results provide a platform for further studies into the molecular machinery of PGC migration in yellowtail kingfish, as well as other perciform fish species.

Factors associated with empyema in children with community acquired pneumonia.

AIM: Empyema is a potential complication of community acquired pneumonia but factors predicting this complication are lacking. METHODS: A retrospective study of all previously healthy pediatric patients admitted between January 2007 and July 2009 with CAP. Patients with non-lobar pneumonia, RSV bronchiolitis, underlying chronic disease, or hospital-acquired pneumonia were excluded. Preadmission, clinical characteristics on admission, and outcome were compared between patients with and without empyema. Management strategies in patients with empyema were also compared. RESULTS: Overall 356 patients were included. Median age was 3.8 ± 3.54 years and 60.7% were males. A total of 43 patients (12%) were diagnosed with empyema. The development of empyema was independently associated, on multivariate analysis, with older age, female gender and antibiotic therapy prior to admission, and with dyspnea, thrombocytopenia and involvement of more than one lobe on chest radiograph on admission. Patients who developed empyema had a longer and more complicated course. Hypoxemia on admission was significantly less frequent in patients with empyema who were treated with antibiotic therapy alone, compared to those treated with chest tube or video-assisted thoracoscopic surgery. CONCLUSION: Early identification of dyspnea and thrombocytopenia in patients with community acquired pneumonia could alert physicians on the potential development of empyema. Antibiotic therapy alone may be sufficient in patients with empyema who are mildly hypoxemic on admission.

In silico analysis of the regulatory region of the Yellowtail Kingfish and Zebrafish Kiss and Kiss receptor genes.

We have cloned and analysed the partial putative promoter sequences of the Yellowtail Kingfish (Seriola lalandi) Kiss2 and Kiss2r genes (380 and 420 bp, respectively). We obtained in silico 1.5 kb of the zebrafish (Danio rerio) Kiss1, Kiss2, Kiss1r and zfKiss2r sequences upstream of the putative transcriptional initiation site. Bioinformatic analysis revealed promoter regulatory elements including AP-1, Sp1, GR, ER, PR, AR, GATA-1, TTF-1, YY1 and C/EBP. These regulatory elements may mediate novel roles of the Kiss genes and their receptors in addition to their established role in reproductive function.

The Kiss2 receptor (Kiss2r) gene in Southern Bluefin Tuna, Thunnus maccoyii and in Yellowtail Kingfish, Seriola lalandi - functional analysis and isolation of transcript variants.

The kisspeptin system plays an essential role in reproductive function in vertebrates, particularly in the onset of puberty. We investigated the kisspeptin system in two Perciform teleosts, the Southern Bluefin Tuna (SBT; Thunnus maccoyii), and the Yellowtail Kingfish (YTK; Seriola lalandi), by characterising their kisspeptin 2 receptor (Kiss2r) genes. In addition to the full length Kiss2r cDNA sequences, we have isolated from SBT and YTK a transcript variant that retained an intron. We have further obtained three ytkKiss2r transcript variants that contained deletions. In vitro functional analysis of the full length SBT and YTK Kiss2r showed higher response to Kiss2-10 than to Kiss1-10, with stronger transduction via PKC than PKA. The full length ytkKiss2r and two deletion variants were differentially expressed in the brain of male, but not in female, juvenile YTK treated with increasing doses of Kiss2-10 peptide. In the gonads, the expression level of the ytkKiss2r transcripts did not vary significantly either in the male or female fish. This is the first time that transcript variants of the Kiss2r gene that contain deletions and show responsiveness to treatments with kisspeptin have been reported in any teleost.

Lung transplantation in infants and toddlers from 1990 to 2004 at St. Louis Children's Hospital.

In a retrospective, single-center cohort study, outcomes of infants and toddlers undergoing lung transplant at St. Louis Children's Hospital between 1990 and 2004 were compared to older children. Patients with cystic fibrosis (exclusively older children) and those who underwent heart-lung, liver-lung, single lung or a second transplantation were excluded from comparisons. One hundred nine lung transplants were compared. Thirty-six were in infants <1 year old, 26 in toddlers 1-3 years old and 47 in children >3 years old. Graft survival was similar for infants and toddlers (p = 0.35 and p = 0.3, respectively) compared to children over 3 years old at 1 and 3 years after transplant. Significantly more infants (p < 0.0001 and p = 0.003) and toddlers (p = 0.002 and p = 0.03) were free from acute rejection and bronchiolitis obliterans compared to older patients. While most infants and toddlers had only minimal lung function impairment, and achieved normal to mildly delayed developmental scores, somatic growth remained depressed 5 years after transplant. Lung transplantation in infants and young children carries similar survival rates to older children and adults. Further insights into the unique immunologic aspects of this group of patients may elucidate strategies to prevent acute and chronic rejection in all age groups.

Sex change strategy and the aromatase genes.

Sequential hermaphroditism is a common reproductive strategy in many teleosts. Steroid production is known to mediate both the natural and induced sex change, yet beyond this the physiology directing this process has received little attention. Cytochrome P450 aromatase is a key enzyme in the hormonal pathway catalysing the conversion of sex steroids, androgens to oestrogens, and thus is highly relevant to the process of sex change. This study reports the isolation of cDNA sequences for aromatase isoforms CYP19A1 and CYP19A2 from teleost species representing three forms of sexual hermaphroditism: Lates calcarifer (protandry), Cromileptes altivelis (protogyny), and Gobiodon histrio (bi-directional). Deduced amino acid analysis of these isoforms with other reported isoforms from gonochoristic (single sex) teleosts revealed 56-95% identity within the same isoform while only 48-65% identity between isoforms irrespective of species and sexual strategy. Phylogenetic analysis supported this result separating sequences into isoform exclusive clades in spite of species apparent evolutionary distance. Furthermore, this study isolates 5' flanking regions of all above genes and describes putative cis-acting elements therein. Elements identified include steroidogenic factor 1 binding site (SF-1), oestrogen response element (ERE), progesterone response element (PRE), androgen response element (ARE), glucocorticoid response elements (GRE), peroxisome proliferator-activated receptor alpha/retinoid X receptor alpha heterodimer responsive element (PPARalpha/RXRalpha), nuclear factor kappabeta (NF-kappabeta), SOX 5, SOX 9, and Wilms tumor suppressor (WTI). A hypothetical in vivo model was constructed for both isoforms highlighting potential roles of these putative cis-acting elements with reference to normal function and sexual hermaphroditism.

Isolation of dopamine D(2) receptor (D (2)R) promoters in Mugil cephalus.

This paper reports the isolation of two putative D(2)R promoters from grey mullet, one 5'flanking and the other an intronic sequence immediately upstream of the first coding exon. Promoter activity of the intronic sequence was confirmed in vitro through functional analysis using luciferase as reporter gene. The functional characteristics of the region flanking the 5'UTR is currently under investigation.

[Representing patients hospitalized under legal commitment at district psychiatric committees].

The Tel Aviv-Central District Office of the Public Defenders' Office has begun, as a trial project, to represent patients hospitalized under enforced commitments at district psychiatric committee hearings concerning their welfare. This experimental trial was carried out at the Abarbanel Mental Health Center starting 1st January 2000. The results illustrate that the chances of a patient hospitalized under enforced commitment being discharged from hospital are better if he is represented. It is particularly better when cooperation exists between his public defender and his treating psychiatrist. The results emphasize that the inclusion of the defender in the process affects the work program of the committee and its decisions, especially the examination of facts, the hearing of witnesses, the quality of the psychiatric assessment, the examination of the legal aspects and the summing up of the findings. From feedback received, it appears that all parties involved in the process feel that representation on behalf of the patients by the legal defenders results in more just and worthy decisions in every public aspect of the process. The authors recommend that legal representation be extended to all patients who are hospitalized under enforced commitments.

Gonadotropin response to GnRH during sexual ontogeny in the common carp, Cyprinus carpio.

This study was designed to reveal whether gonadotropic response to GnRH in the common carp (Cyprinus carpio) changes during sexual ontogeny and whether the response of FSHbeta and LHbeta subunits is uniform or differential. The study comprised fish at the following stages: juveniles (4-month-old females with primary oocytes and early spermatogenic males); maturing (9-month-old previtellogenic females and advanced spermatogenic males); and mature (16-month-old postvitellogenic females and spermiating males). Fish were injected with superactive salmon GnRH analogue (sGnRHa; 25 microg/kg) and blood was sampled 6, 12 and 24 h later for cGtH (LH) and sex steroid levels. Pituitaries were taken for determination of FSHbeta and LHbeta mRNA levels by slot-blot hybridization and for cGTH content in the same glands by radioimmunoassay (RIA). Values were compared with the levels prior to sGnRHa administration and with control fish sampled at the same intervals. Juvenile fish did not respond at all to sGnRHa. In maturing females, FSHbeta mRNA increased by >300%, while that of LHbeta increased by 200%. In maturing males, FSHbeta mRNA did not change and only a slight increase occurred in that of LHbeta. In 16-month-old postvitellogenic females, there was no response of FSHbeta mRNA, while that of LHbeta dramatically increased. In spermiating males of the same age, mRNA of both FSHbeta and LHbeta increased following sGnRHa injection. Immunoreactive cGtH was present in the pituitary and plasma of all fish examined, but in juveniles it did not change following sGnRHa injection. In maturing and mature fish of both genders, sGnRHa administration was followed by a marked increase in circulating cGtH, concomitant with a decrease in its pituitary content, indicating the limited amount of the hormone stored in the gland. In conclusion, the response of the gonadotropin subunit mRNAs in the common carp was found to be differential and dependent on the gender and the phase of sexual ontogeny.

The GnRH system in the European sea bass (Dicentrarchus labrax).

The cDNA sequences encoding three GnRH forms, sea bream GnRH (sbGnRH), salmon GnRH (sGnRH) and chicken GnRH II (cGnRH II), were cloned from the brain of European sea bass, Dicentrarchus labrax. Comparison of their deduced amino acid sequences to the same forms in the gilthead sea bream, Sparus aurata, and striped bass, Morone saxatilis, revealed high homology of the prepro-cGnRH II (94% and 98% respectively), and prepro-sGnRH (92% to both species). The sbGnRH exhibited dissimilar identities, with high homology to the striped bass (93%), and lower homology (59%) to the gilthead sea bream. Two transcript types were identified for the GnRH-associated peptide (GAP)-sGnRH as well as for the GAP-cGnRH II, which suggests a possible alternative splicing followed by the addition of an early stop codon. In order to obtain antibodies specific for the three GnRH precursors, recombinant GAP proteins were produced. The differential expression of the three GnRHs previously reported in the brain by means of in situ hybridization, using riboprobes corresponding to the GAP-coding regions, was fully confirmed by immunocytochemistry using antibodies raised against the recombinant GAP proteins, indicating that the transcripts are translated into functional proteins. Moreover, this approach allowed us to follow, for the first time, the specific projections of the different cell groups: sGAP fibers are distributed mainly in the forebrain with few projections reaching the pituitary, sbGAP fibers are mainly present in the preoptic area, mediobasal hypothalamus and predominantly project to the pars distalis of the pituitary, whereas cGnRH II fibers have a widespread distribution primarily in the posterior brain, and do not project to the pituitary. These new tools will be extremely useful to study further the development, regulation and functional significance of three independent GnRH systems in the brain of vertebrate species.

Melatonin treatment for tardive dyskinesia: a double-blind, placebo-controlled, crossover study.

BACKGROUND: Antipsychotics remain the mainstay of drug intervention in the management of schizophrenia. However, long-term treatment with antipsychotics is associated with a variety of movement disorders, the most disabling of which is tardive dyskinesia (TD), which occurs in up to 50% of patients hospitalized with chronic schizophrenia. The pathophysiology of TD is still unclear and no definite treatment exists. Both dopamine receptor supersensitivity and oxidative stress-induced neurotoxicity in the nigrostriatal system are apparently implicated. The pineal hormone melatonin is a potent antioxidant and attenuates dopaminergic activity in the striatum and dopamine release from the hypothalamus. Thus, it may have a beneficial effect for both the treatment and prevention of TD. METHODS: Using a double-blind, placebo-controlled, crossover study, we evaluated the efficacy of 10 mg/d of melatonin for 6 weeks in 22 patients with schizophrenia and TD. The primary outcome measure was the change from baseline in Abnormal Involuntary Movement Scale (AIMS) score. RESULTS: The decrease (mean +/- SD) in AIMS score was 2.45 +/- 1.92 for the melatonin and 0.77 +/- 1.11 for the placebo treatment groups (P<.001). No adverse events or side effects were noted. CONCLUSION: This is the first clinical evidence for efficacy of melatonin in the treatment of TD.

Retinol binding protein in rainbow trout: molecular properties and mRNA expression in tissues.

Retinoids are important regulatory signaling molecules during embryonic development. The molecular properties of rainbow trout (Oncorhynchus mykiss) retinol-binding protein (rtRBP), the specific retinol carrier in vertebrate plasma, were studied to elucidate its role in transporting retinols to developing fish oocytes. A 954-nucleotide rtRBP cDNA was cloned from the liver coding for a 176-amino-acid (aa) mature protein, with an estimated molecular mass of 20,267 Da. The nucleotide sequence suggests a putative 16-aa signal peptide and shows all the aa residues that were previously identified as critical for the retinol binding pocket. Five of the eight amino acid residues that are associated with the interaction of RBP and transthyretin in mammalian and non-mammalian species are conserved. The deduced aa sequence of rtRBP shows 60-66% identity with zebrafish, chicken, mouse, rat, horse, bovine, and human RBPs and 56% identity with Xenopus RBP. Northern blot analysis revealed a approximately 1.1-kb hepatic mRNA transcript. RBP is highly expressed in the liver, but low levels were also detected in the spleen, kidney, ovary, and brain. In the rainbow trout, 17beta-estradiol treatment led to a decrease in the RBP mRNA signal relative to that of the controls. The efficacy of the 17beta-estradiol treatment was verified by an induction of vitellogenin (VTG) mRNA expression in the liver and occurrence of VTG in the plasma.

Tilapia glycoprotein hormone alpha subunit: cDNA cloning and hypothalamic regulation.

The cDNA encoding the glycoprotein alpha (GPalpha) subunit of tilapia (Oreochromis mossambicus) was partially cloned using RACE-polymerase chain reaction (PCR) technique. The amplified cDNA was found to be 583 bases long, and to consist of a portion of the signal peptide, the full sequence encoding the mature peptide (94 amino acids) and the 3' untranslated region. Northern blot analysis revealed a single band of approximately 600 bp. Alignment of the deduced amino acids of the mature protein showed that the tilapia GPalpha subunit shares more than 80% identity with that of other perciform fish (i.e. striped bass, sea bream and yellowfin porgy) and less than 70% with that of more taxonomically remote fish and other vertebrates. Exposure of dispersed tilapia pituitary cells to salmon gonadotropin-releasing hormone (sGnRH) elevated GPalpha mRNA levels via both PKC and cAMP-protein kinase A (PKA) pathways. The transcript levels were also regulated by pituitary adenylate cyclase activating polypeptide (PACAP) and neuropeptide Y (NPY), both acting through PKC and PKA pathways. Moreover, a combined treatment of PACAP or NPY with GnRH seems to have an additive effect on the GPalpha subunit gene transcription. These results suggest that in tilapia the expression of GPalpha subunit is regulated by GnRH mainly via PKC and PKA pathways. Furthermore, PACAP and NPY can elevate the GnRH-stimulated GPalpha subunit transcription and can directly affect the subunit mRNA levels, via the same transduction pathways.