Pharmacological and behavioural effects of tryptamines present in psilocybin-containing mushrooms.

BACKGROUND AND PURPOSE: Demand for new antidepressants has resulted in a re-evaluation of the therapeutic potential of psychedelic drugs. Several tryptamines found in psilocybin-containing "magic" mushrooms share chemical similarities with psilocybin. Early work suggests they may share biological targets. However, few studies have explored their pharmacological and behavioural effects. EXPERIMENTAL APPROACH: We compared baeocystin, norbaeocystin and aeruginascin with psilocybin to determine if they are metabolized by the same enzymes, similarly penetrate the blood-brain barrier, serve as ligands for similar receptors and modulate behaviour in rodents similarly. We also assessed the stability and optimal storage and handling conditions for each compound. KEY RESULTS: In vitro enzyme kinetics assays found that all compounds had nearly identical rates of dephosphorylation via alkaline phosphatase and metabolism by monoamine oxidase. Further, we found that only the dephosphorylated products of baeocystin and norbaeocystin crossed a blood-brain barrier mimetic to a similar degree as the dephosphorylated form of psilocybin, psilocin. The dephosphorylated form of norbaeocystin was found to activate the 5-HT2A receptor with similar efficacy to psilocin and norpsilocin in in vitro cell imaging assays. Behaviourally, only psilocybin induced head twitch responses in rats, a marker of 5-HT2A-mediated psychedelic effects and hallucinogenic potential. However, like psilocybin, norbaeocystin improved outcomes in the forced swim test. All compounds caused minimal changes to metrics of renal and hepatic health, suggesting innocuous safety profiles. CONCLUSIONS AND IMPLICATIONS: Collectively, this work suggests that other naturally occurring tryptamines, especially norbaeocystin, may share overlapping therapeutic potential with psilocybin, but without causing hallucinations.

Evaluation of TrpM and PsiD substrate promiscuity reveals new biocatalytic capabilities.

N-methylated tryptamines, such as the hallucinogenic natural products, psilocybin and N,N-dimethyltryptamine (DMT), are gaining interest from the medical community due to their potential as next generation treatments for mental health disorders. The clinical relevance of these compounds has driven scientists to develop biosynthetic production routes to a number of tryptamine drug candidates, and efforts are ongoing to expand and further develop these biosynthetic capabilities. To that end, we have further characterized the substrate preferences of two enzymes involved in tryptamine biosynthesis: TrpM, a tryptophan N-methyltransferase from Psilocybe serbica, and PsiD, the gateway decarboxylase of the psilocybin biosynthesis pathway. Here, we show that TrpM can N-methylate the non-native amino acid substrate, 4-hydroxytryptophan, a key intermediate in the Escherichia coli-based recombinant psilocybin biosynthesis pathway. However, the ability to incorporate TrpM into a functional psilocybin biosynthesis pathway was thwarted by PsiD's inability to use N,N-dimethyl-4-hydroxytryptophan as substrate, under the culturing conditions tested, despite demonstrating activity on N-methylated and 4-hydroxylated tryptophan derivatives individually. Taken together, this work expands upon the known substrates for TrpM and PsiD, further increasing the diversity of tryptamine biosynthetic products.

"Biosynthesis of psilocybin and its nonnatural derivatives by a promiscuous psilocybin synthesis pathway in Escherichia coli".

Traditional psychedelics are undergoing a transformation from recreational drugs, to promising pharmaceutical drug candidates with the potential to provide an alternative treatment option for individuals struggling with mental illness. Sustainable and economic production methods are thus needed to facilitate enhanced study of these drug candidates to support future clinical efforts. Here, we expand upon current bacterial psilocybin biosynthesis by incorporating the cytochrome P450 monooxygenase, PsiH, to enable the de novo production of psilocybin as well as the biosynthesis of 13 psilocybin derivatives. The substrate promiscuity of the psilocybin biosynthesis pathway was comprehensively probed by using a library of 49 single-substituted indole derivatives, providing biophysical insights to this understudied metabolic pathway and opening the door to the in vivo biological synthesis of a library of previously unstudied pharmaceutical drug candidates.

"In vivo biosynthesis of N,N-dimethyltryptamine, 5-MeO-N,N-dimethyltryptamine, and bufotenine in E.coli".

N,N-dimethyltryptamine (DMT), 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) and 5-hydroxy-N,N-dimethyltryptamine (bufotenine) are psychedelic tryptamines found naturally in both plants and animals and have shown clinical potential to help treat mental disorders, such as anxiety and depression. Advances in both metabolic and genetic engineering make it possible to engineer microbes as cell factories to produce DMT and its aforementioned derivatives to meet demand for ongoing clinical study. Here, we present the development of a biosynthetic production pathway for DMT, 5-MeO-DMT, and bufotenine in the model microbe Escherichia coli. Through the application of genetic optimization techniques and process optimization in benchtop fermenters, the in vivo production of DMT in E. coli was observed. DMT production with tryptophan supplementation reached maximum titers of 74.7 ± 10.5 mg/L under fed batch conditions in a 2-L bioreactor. Additionally, we show the first reported case of de novo production of DMT (from glucose) in E. coli at a maximum titer of 14.0 mg/L and report the first example of microbial 5-MeO-DMT and bufotenine production in vivo. This work provides a starting point for further genetic and fermentation optimization studies with the goal to increase methylated tryptamine production metrics to industrially competitive levels.

Effect of psilocybin on decision-making and motivation in the healthy rat.

Psilocybin and its active metabolite psilocin are hallucinogenic serotonergic agonists with high affinity for several serotonin receptors. In addition to underlying the hallucinogenic effects of these compounds, serotonin receptor activation also has important effects on decision-making and goal-directed behaviors. The impact of psilocybin and psilocin on these cognitive systems, however, remains unclear. This study investigated the effects of psilocybin treatment on decision-making and motivation in healthy male and female rats. We compared probability and delay discounting performance of psilocybin treated (1 mg/kg) to vehicle rats (n = 10/sex/group), and further assessed motivation in each group using a progressive ratio task. We also confirmed drug action by assessing head twitch responses after psilocybin treatment (1 mg/kg). Results from this study demonstrated that exposure to 1 mg/kg psilocybin did not affect decision-making in the probability and delay discounting tasks and did not reduce response rates in the progressive ratio task. However, psilocybin treatment did cause the expected increase in head twitch responses in both male and female rats, demonstrating that the drug was delivered at a pharmacologically relevant dosage. Combined, these results suggest that psilocybin may not impair or improve decision-making and motivation. Considering recent interest in psilocybin as a potential fast-acting therapeutic for a variety of mental health disorders, our findings also suggest the therapeutic effects of this drug may not be mediated by changes to the brain systems underlying reward and decision-making. Finally, these results may have important implications regarding the relative safety of this compound, suggesting that widespread cognitive impairments may not be seen in subjects, even after chronic treatment.

Development of an E. coli-based norbaeocystin production platform and evaluation of behavioral effects in rats.

Interest in the potential therapeutic efficacy of psilocybin and other psychedelic compounds has escalated significantly in recent years. To date, little is known regarding the biological activity of the psilocybin pathway intermediate, norbaeocystin, due to limitations around sourcing the phosphorylated tryptamine metabolite for in vivo testing. To address this limitation, we first developed a novel E. coli platform for the rapid and scalable production of gram-scale amounts of norbaeocystin. Through this process we compare the genetic and fermentation optimization strategies to that of a similarly constructed and previously reported psilocybin producing strain, uncovering the need for reoptimization and balancing upon even minor genetic modifications to the production host. We then perform in vivo measurements of head twitch response to both biosynthesized psilocybin and norbaeocystin using both a cell broth and water vehicle in Long-Evans rats. The data show a dose response to psilocybin while norbaeocystin does not elicit any pharmacological response, suggesting that norbaeocystin and its metabolites may not have a strong affinity for the serotonin 2A receptor. The findings presented here provide a mechanism to source norbaeocystin for future studies to evaluate its disease efficacy in animal models, both individually and in combination with psilocybin, and support the safety of cell broth as a drug delivery vehicle.

Homebrewed psilocybin: can new routes for pharmaceutical psilocybin production enable recreational use?

Psilocybin, a drug most commonly recognized as a recreational psychedelic, is quickly gaining attention as a promising therapy for an expanding range of neurological conditions, including depression, anxiety, and addiction. This growing interest has led to many recent advancements in psilocybin synthesis strategies, including multiple in vivo fermentation-based approaches catalyzed by recombinant microorganisms. In this work, we show that psilocybin can be produced in biologically relevant quantities using a recombinant E. coli strain in a homebrew style environment. In less than 2 days, we successfully produced approximately 300 mg/L of psilocybin under simple conditions with easily sourced equipment and supplies. This finding raises the question of how this new technology should be regulated as to not facilitate clandestine biosynthesis efforts, while still enabling advancements in psilocybin synthesis technology for pharmaceutical applications. Here, we present our homebrew results, and suggestions on how to address the regulatory concerns accompanying this new technology.

Gpr63 is a modifier of microcephaly in Ttc21b mouse mutants.

The primary cilium is a signaling center critical for proper embryonic development. Previous studies have demonstrated that mice lacking Ttc21b have impaired retrograde trafficking within the cilium and multiple organogenesis phenotypes, including microcephaly. Interestingly, the severity of the microcephaly in Ttc21baln/aln homozygous null mutants is considerably affected by the genetic background and mutants on an FVB/NJ (FVB) background develop a forebrain significantly smaller than mutants on a C57BL/6J (B6) background. We performed a Quantitative Trait Locus (QTL) analysis to identify potential genetic modifiers and identified two regions linked to differential forebrain size: modifier of alien QTL1 (Moaq1) on chromosome 4 at 27.8 Mb and Moaq2 on chromosome 6 at 93.6 Mb. These QTLs were validated by constructing congenic strains. Further analysis of Moaq1 identified an orphan G-protein coupled receptor (GPCR), Gpr63, as a candidate gene. We identified a SNP that is polymorphic between the FVB and B6 strains in Gpr63 and creates a missense mutation predicted to be deleterious in the FVB protein. We used CRISPR-Cas9 genome editing to create two lines of FVB congenic mice: one with the B6 sequence of Gpr63 and the other with a deletion allele leading to a truncation of the GPR63 C-terminal tail. We then demonstrated that Gpr63 can localize to the cilium in vitro. These alleles affect ciliary localization of GPR63 in vitro and genetically interact with Ttc21baln/aln as Gpr63;Ttc21b double mutants show unique phenotypes including spina bifida aperta and earlier embryonic lethality. This validated Gpr63 as a modifier of multiple Ttc21b neural phenotypes and strongly supports Gpr63 as a causal gene (i.e., a quantitative trait gene, QTG) within the Moaq1 QTL.

Heart disease in a mutant mouse model of spontaneous eosinophilic myocarditis maps to three loci.

BACKGROUND: Heart disease (HD) is the major cause of morbidity and mortality in patients with hypereosinophilic diseases. Due to a lack of adequate animal models, our understanding of the pathophysiology of eosinophil-mediated diseases with heart complications is limited. We have discovered a mouse mutant, now maintained on an A/J inbred background, that spontaneously develops hypereosinophilia in multiple organs. Cellular infiltration into the heart causes an eosinophilic myocarditis, with affected mice of the mutant line (i.e., A/JHD) demonstrating extensive myocardial damage and remodeling that leads to HD and premature death, usually by 15-weeks old. RESULTS: Maintaining the A/JHD line for many generations established that the HD trait was heritable and implied the mode of inheritance was not too complex. Backcross and intercross populations generated from mating A/JHD males with females from four different inbred strains produced recombinant populations with highly variable rates of affected offspring, ranging from none in C57BL/6 J intercrosses, to a few mice with HD using 129S1/SvImJ intercrosses and C57BL/6 J backcrosses, but nearly 8% of intercrosses and > 17% of backcrosses from SJL/J related populations developed HD. Linkage analyses of these SJL/J derived recombinants identified three highly significant loci: a recessive locus mapping to distal chromosome 5 (LOD = 4.88; named Emhd1 for eosinophilic myocarditis to heart disease-1); and two dominant variants mapping to chromosome 17, one (Emhd2; LOD = 7.51) proximal to the major histocompatibility complex, and a second (Emhd3; LOD = 6.89) that includes the major histocompatibility region. Haplotype analysis identified the specific crossovers that defined the Emhd1 (2.65 Mb), Emhd2 (8.46 Mb) and Emhd3 (14.59 Mb) intervals. CONCLUSIONS: These results indicate the HD trait in this mutant mouse model of eosinophilic myocarditis is oligogenic with variable penetrance, due to multiple segregating variants and possibly additional genetic or nongenetic factors. The A/JHD mouse model represents a unique and valuable resource to understand the interplay of causal factors that underlie the pathology of this newly discovered eosinophil-associated disease with cardiac complications.

Characterization of a mouse model of hypereosinophilia-associated heart disease.

Hypereosinophilic syndrome is characterized by sustained and marked eosinophilia leading to tissue damage and organ dysfunction. Morbidity and mortality occur primarily due to cardiac and thromboembolic complications. Understanding the cause and mechanism of disease would aid in the development of targeted therapies with greater efficacy and fewer side effects. We discovered a spontaneous mouse mutant in our colony with a hypereosinophilic phenotype. Mice develop peripheral blood eosinophilia; infiltration of lungs, spleen, and heart by eosinophils; and extensive myocardial damage and remodeling. This ultimately leads to heart failure and premature death. Histopathological assessment of the hearts revealed a robust inflammatory infiltrate composed primarily of eosinophils and B-lymphocytes, associated with myocardial damage and replacement fibrosis, consistent with eosinophilic myocarditis. In many cases, hearts showed dilatation and thinning of the right ventricular wall, suggestive of an inflammatory dilated cardiomyopathy. Most mice showed atrial thrombi, which often filled the chamber. Protein expression analysis revealed overexpression of chemokines and cytokines involved in innate and adaptive immunity including IL-4, eotaxin, and RANTES. Disease could be transferred to wild-type mice by adoptive transfer of splenocytes from affected mice, suggesting a role for the immune system. In summary, the pathologies observed in the mutant lines are reminiscent of those seen in patients with hypereosinophilia, where cardiac-related morbidities, like congestive heart failure and thrombi, are the most common causes of death. As such, our model provides an opportunity to test mechanistic hypotheses and develop targeted therapies.NEW & NOTEWORTHY This article describes a new model of heart disease in hypereosinophilia. The model developed as a spontaneous mouse mutant in the colony and is characterized by peripheral blood eosinophilia and infiltration of lungs, spleen, and heart by eosinophils. In the heart, there is extensive myocardial damage, remodeling, fibrosis, and thrombosis, leading to heart failure and death. The immune microenvironment is one of increased innate and adaptive immunity, including Th1 and Th2 cytokines/chemokines. Finally, adoptive transfer of splenocytes transfers disease to recipient mice. In summary, this model provides an opportunity to test mechanistic hypotheses and develop targeted therapies for this rare but devastating disease.

Iatrogenic "buffalo chest" bilateral pneumothoraces following unilateral transbronchial lung biopsies in a bilateral lung transplant recipient.

We present a 54 year old male patient who had a bilateral lung transplant sixteen years ago for Alpha-1 Antitrypsin Deficiency-related emphysema. He was referred for flexible bronchoscopy with transbronchial biopsies to evaluate new mild exertional dyspnea and worsening of his FEV1. Eight transbronchial biopsies were done from the right middle lobe and the right lower lobe. Post procedure he developed bilateral pneumothoces that required emergent bilateral pleural 'pigtail' catheters. To our knowledge, this is the first reported case of bilateral pneumothoraces that developed after a unilateral procedure in a bilateral lung transplant recipient relatively late after the transplant.

Age and Sex of Mice Markedly Affect Survival Times Associated with Hyperoxic Acute Lung Injury.

Mortality associated with acute lung injury (ALI) remains substantial, with recent estimates of 35-45% similar to those obtained decades ago. Although evidence for sex-related differences in ALI mortality remains equivocal, death rates differ markedly for age, with more than 3-fold increased mortality in older versus younger patients. Strains of mice also show large differences in ALI mortality. To tease out genetic factors affecting mortality, we established a mouse model of differential hyperoxic ALI (HALI) survival. Separate genetic analyses of backcross and F2 populations generated from sensitive C57BL/6J (B) and resistant 129X1/SvJ (X1) progenitor strains identified two quantitative trait loci (QTLs; Shali1 and Shali2) with strong, equal but opposite, within-strain effects on survival. Congenic lines confirmed these opposing QTL effects, but also retained the low penetrance seen in the 6-12 week X1 control strain. Sorting mice into distinct age groups revealed that 'age at exposure' inversely correlated with survival time and explained reduced penetrance of the resistance trait. While B mice were already sensitive by 6 weeks old, X1 mice maintained significant resistance up to 3-4 weeks longer. Reanalysis of F2 data gave analogous age-related findings, and also supported sex-specific linkage for Shali1 and Shali2. Importantly, we have demonstrated in congenic mice that these age effects on survival correspond with B alleles for Shali1 (6-week old mice more sensitive) and Shali2 (10-week old mice more resistant) placed on the X1 background. Further studies revealed significant sex-specific survival differences in subcongenics for both QTLs. Accounting for age and sex markedly improved penetrance of both QTLs, thereby reducing trait variability, refining Shali1 to <8.5Mb, and supporting several sub-QTLs within the Shali2 interval. Together, these congenics will allow age- and sex-specific studies to interrogate myriad subphenotypes affected during ALI development and progression and identify intermediary injury biomarkers that can predict outcome.

Synchronizing allelic effects of opposing quantitative trait loci confirmed a major epistatic interaction affecting acute lung injury survival in mice.

Increased oxygen (O(2)) levels help manage severely injured patients, but too much for too long can cause acute lung injury (ALI), acute respiratory distress syndrome (ARDS) and even death. In fact, continuous hyperoxia has become a prototype in rodents to mimic salient clinical and pathological characteristics of ALI/ARDS. To identify genes affecting hyperoxia-induced ALI (HALI), we previously established a mouse model of differential susceptibility. Genetic analysis of backcross and F(2) populations derived from sensitive (C57BL/6J; B) and resistant (129X1/SvJ; X1) inbred strains identified five quantitative trait loci (QTLs; Shali1-5) linked to HALI survival time. Interestingly, analysis of these recombinant populations supported opposite within-strain effects on survival for the two major-effect QTLs. Whereas Shali1 alleles imparted the expected survival time effects (i.e., X1 alleles increased HALI resistance and B alleles increased sensitivity), the allelic effects of Shali2 were reversed (i.e., X1 alleles increased HALI sensitivity and B alleles increased resistance). For in vivo validation of these inverse allelic effects, we constructed reciprocal congenic lines to synchronize the sensitivity or resistance alleles of Shali1 and Shali2 within the same strain. Specifically, B-derived Shali1 or Shali2 QTL regions were transferred to X1 mice and X1-derived QTL segments were transferred to B mice. Our previous QTL results predicted that substituting Shali1 B alleles onto the resistant X1 background would add sensitivity. Surprisingly, not only were these mice more sensitive than the resistant X1 strain, they were more sensitive than the sensitive B strain. In stark contrast, substituting the Shali2 interval from the sensitive B strain onto the X1 background markedly increased the survival time. Reciprocal congenic lines confirmed the opposing allelic effects of Shali1 and Shali2 on HALI survival time and provide unique models to identify their respective quantitative trait genes and to critically assess the apparent bidirectional epistatic interactions between these major-effect loci.

Genomewide association analysis of respiratory syncytial virus infection in mice.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in infants, with about half being infected in their first year of life. Yet only 2 to 3% of infants are hospitalized for RSV infection, suggesting that individual susceptibility contributes to disease severity. Previously, we determined that AKR/J (susceptible) mice developed high lung RSV titers and showed delayed weight recovery, whereas C57BL/6J (resistant) mice demonstrated low lung RSV titers and rapid weight recovery. In addition, we have reported that gene-targeted mice lacking the cystic fibrosis transmembrane conductance regulator (Cftr; ATP-binding cassette subfamily C, member 7) are susceptible to RSV infection. For this report, recombinant backcross and F2 progeny derived from C57BL/6J and AKR/J mice were infected with RSV, their lung titers were measured, and quantitative trait locus (QTL) analysis was performed. A major QTL, designated Rsvs1, was identified on proximal mouse chromosome 6 in both recombinant populations. Microarray analysis comparing lung transcripts of the parental strains during infection identified several candidate genes that mapped to the Rsvs1 interval, including Cftr. These findings add to our understanding of individual RSV susceptibility and strongly support a modifier role for CFTR in RSV infection, a significant cause of respiratory morbidity in infants with cystic fibrosis.

Reciprocal backcross mice confirm major loci linked to hyperoxic acute lung injury survival time.

Morbidity and mortality associated with acute lung injury (ALI) and acute respiratory distress syndrome remain substantial. Although many candidate genes have been tested, a clear understanding of the pathogenesis is lacking, as is our ability to predict individual outcome. Because ALI is a complex disease, single gene approaches cannot easily identify effectors that must be treated concurrently. We employed a strategy to help identify critical genes and gene combinations involved in ALI mortality. Using hyperoxia to induce ALI, a mouse model for genetic analyses of ALI survival time was identified: C57BL/6J (B) mice are sensitive (i.e., die early), whereas 129X1/SvJ (S) mice are significantly more resistant, but with low penetrance. Segregation analysis of reciprocal F(2) mice generated from B and S strains revealed significant sex, cross, and parent of origin effects. Quantitative trait locus (QTL) analysis identified five chromosomal regions significantly linked to hyperoxic ALI survival time (named Shali1-Shali5). Further analyses demonstrated that both parental strains contribute resistance alleles to their offspring and that the phenotype demonstrated parent of origin effects. To validate earlier findings, we generated and tested mice from all eight possible B-S-derived backcrosses. Results from segregation and QTL analyses of 935 backcrosses, alone and combined with the previous 840 B-S-derived F(2) population, further supported the highly significant QTLs on chromosomes 1 (Shali1) and 4 (Shali2) and confirmed that the sex, cross, and parent of origin all contribute to survival time with hyperoxic ALI.

Reciprocal congenic lines of mice capture the aliq1 effect on acute lung injury survival time.

Acute lung injury (ALI) is a devastating condition resulting from diverse causes. Genetic studies of human populations indicate that ALI is a complex disease with substantial phenotypic variance, incomplete penetrance, and gene-environment interactions. To identify genes controlling ALI mortality, we previously investigated mean survival time (MST) differences between sensitive A/J (A) and resistant C57BL/6J (B) mice in ozone using quantitative trait locus (QTL) analysis. MST was significantly linked to QTLs (Aliq1-3) on chromosomes 11, 13, and 17, respectively. Additional QTL analyses of separate and combined backcross and F(2) populations supported linkage to Aliq1 and Aliq2, and established significance for previously suggestive QTLs on chromosomes 7 and 12 (named Aliq5 and Aliq6, respectively). Decreased MSTs of corresponding chromosome substitution strains (CSSs) verified the contribution of most QTL-containing chromosomes to ALI survival. Multilocus models demonstrated that three QTLs could explain the MST difference between progenitor strains, agreeing with calculated estimates for number of genes involved. Based on results of QTL genotype analysis, a double CSS (B.A-6,11) was generated that contained Aliq1 and Aliq4 chromosomes. Surprisingly, MST and pulmonary edema after exposure of B.A-6,11 mice were comparable to B mice, revealing an unpredicted loss of sensitivity compared with separate CSSs. Reciprocal congenic lines for Aliq1 captured the corresponding phenotype in both background strains and further refined the QTL interval. Together, these findings support most of the previously identified QTLs linked to ALI survival and established lines of mice to further resolve Aliq1.

A genetic mouse model to investigate hyperoxic acute lung injury survival.

Acute lung injury (ALI) is a devastating disease that maintains a high mortality rate, despite decades of research. Hyperoxia, a universal treatment for ALI and other critically ill patients, can itself cause pulmonary damage, which drastically restricts its therapeutic potential. We stipulate that having the ability to use higher levels of supplemental O2 for longer periods would improve recovery rates. Toward this goal, a mouse model was sought to identify genes contributing to hyperoxic ALI (HALI) mortality. Eighteen inbred mouse strains were screened in continuous >95% O2. A significant survival difference was identified between sensitive C57BL/6J and resistant 129X1/SvJ strains. Although resistant, only one-fourth of 129X1/SvJ mice survived longer than any C57BL/6J mouse, demonstrating decreased penetrance of resistance. A survival time difference between reciprocal F1 mice implicated a parent-of-origin (imprinting) effect. To further evaluate imprinting and begin to delineate the genetic components of HALI survival, we generated and phenotyped offspring from all four possible intercrosses. Segregation analysis supported maternal inheritance of one or more genes but paternal inheritance of one or more contributor genes. A significant sex effect was demonstrated, with males more resistant than females for all F2 crosses. Survival time ranges and sensitive-to-resistant ratios of the different F2 crosses also supported imprinting and predicted that increased survival is due to dominant resistance alleles contributed by both the resistant and sensitive parental strains. HALI survival is multigenic with a complex mode of inheritance, which should be amenable to genetic dissection with this mouse model.

Genetic analysis of hyperoxic acute lung injury survival in reciprocal intercross mice.

Acute lung injury (ALI) and its most severe presentation, acute respiratory distress syndrome, represent a full spectrum of a complex and devastating illness, with associated mortality that still hovers around 30-40%. Even supplemental O2, a routine and necessary therapy for such patients, paradoxically causes lung injury. The detrimental effects of O2 have established hyperoxic ALI (HALI) as a conventional model to study neonatal and adult forms of respiratory distress syndromes in experimental animals. To confront the high ALI mortality problem quite differently, we recently identified a mouse model (sensitive C57BL/6J and resistant 129X1/SvJ mice) to assess the genetic complexity of HALI and to identify genes affecting strain survival differences. Segregation analysis of 840 F2 mice generated from all four possible intercrosses between C57BL/6J and 129X1/SvJ mice demonstrated that survival time is a quantitative trait with decreased penetrance, and significant sex, cross, and parent-of-origin effects. Quantitative trait locus (QTL) analyses of the total F2 population identified three highly significant (named Shali1, Shali2 and Shali3, for Survival to hyperoxic acute lung injury) and one significant (Shali4) linkage. Analysis of F2 subpopulations further identified a male-specific QTL (Shali5). QTL allelic comparisons supported cross and sex effects and were consistent with imprinting. Genome-wide pairwise analysis predicted additive gene-gene interactions between the QTLs and also revealed a significant epistatic interaction with an otherwise unlinked region. QTL results confirmed that both parental strains contribute dominant resistance alleles to their offspring to determine individual HALI susceptibility.

Development of an unsupported arm exercise test in patients with chronic obstructive pulmonary disease.

BACKGROUND: Unsupported arm exercise tests have been used to evaluate the effects of pulmonary rehabilitation in patients with chronic obstructive pulmonary disease (COPD), but the reliability and validity of these tests are not established. OBJECTIVE: We evaluated the test-retest reliability and validity of a 6-minute pegboard and ring test (PBRT) in 27 outpatients with COPD and 30 age-matched controls. METHODS: We evaluated a 6-minute PBRT, subject demographics, pulmonary function and disease-specific quality of life questionnaire, and the Pulmonary Functional Status Dyspnea Questionnaire-Modified version in 27 patients with COPD. RESULTS: Highly significant correlation coefficients (r = .91, P < .001) were found between test and retest of PBRT scores. Statistically significant correlation coefficients were found between PBRT scores and pulmonary function tests such as FEV1% pred and FVC% pred, and activity domain and subdomain of Pulmonary Functional Status Dyspnea Questionnaire-Modified (P

Solid-state NMR studies of a diverged microsomal amino-proximate delta12 desaturase peptide reveal causes of stability in bilayer: tyrosine anchoring and arginine snorkeling.

This study reports the solid-state NMR spectroscopic characterization of the amino-proximate transmembrane domain (TM-A) of a diverged microsomal delta12-desaturase (CREP-1) in a phospholipid bilayer. A series of TM-A peptides were synthesized with 2H-labeled side chains (Ala-53, -56, and -63, Leu-62, Val-50), and their dynamic properties were studied in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) bilayers at various temperatures. At 6 mol % peptide to lipid, 31P NMR spectra indicated that the peptides did not significantly disrupt the phospholipid bilayer in the L(alpha) phase. The 2H NMR spectra from Ala-53 and Ala-56 samples revealed broad Pake patterns with quadrupolar splittings of 16.9 kHz and 13.3 kHz, respectively, indicating restricted motion confined within the hydrocarbon core of the phospholipid bilayer. Conversely, the deuterated Ala-63 sample revealed a peak centered at 0 kHz with a linewidth of 1.9 kHz, indicating increased side-chain motion and solvent exposure relative to the spectra of the other Ala residues. Val-50 and Leu-62 showed Pake patterns, with quadrupolar splittings of 3.5 kHz and 3.7 kHz, respectively, intermediate to Ala-53/Ala-56 and Ala-63. This indicates partial motional averaging and supports a model with the Val and Leu residues embedded inside the lipid bilayer. Solid-state NMR spectroscopy performed on the 2H-labeled Ala-56 TM-A peptide incorporated into magnetically aligned phospholipid bilayers indicated that the peptide is tilted 8 degrees with respect to the membrane normal of the lipid bilayer. Snorkeling and anchoring interactions of Arg-44 and Tyr-60, respectively, with the polar region or polar hydrophobic interface of the lipid bilayer are suggested as control elements for insertional depth and orientation of the helix in the lipid matrix. Thus, this study defines the location of key residues in TM-A with respect to the lipid bilayer, describes the conformation of TM-A in a biomembrane mimic, presents a peptide-bilayer model useful in the consideration of local protein folding in the microsomal desaturases, and presents a model of arginine and tyrosine control of transmembrane protein stability and insertion.