👤 Robert W Lawrence

Upwards of 50% of people do not respond to the primary treatment modalities for major depressive disorder (MDD), which has led to increased attention and use of alternative methods, including exercise and psychedelics. While interventions using either exercise or psychedelics have demonstrated largely positive results in isolation, their synergistic potential has yet to be explored. As such, this commentary provides an overview of exercise/psychedelics as a treatment for depression and their potential synergy and/or complementarity. From a biological perspective, psychedelics acutely enhance brain-derived neurotrophic factor (BDNF) signalling, while exercise provides sustained BDNF elevation; psychedelics enhance neuroplasticity largely in the cortex (with only modest effects in the hippocampus), while exercise boosts hippocampal neurogenesis; psychedelics increase glutamate release via stimulation of 5-HT Show less

Amphetamine (AMPH) exerts metabolic and cardiovascular effects. The central melanocortin system is a key regulator of both metabolic and cardiovascular functions. Here, we show that the melanocortin system partially mediates AMPH-induced anorexia, energy expenditure, tachycardia, and hypertension. AMPH increased α-melanocyte stimulating hormone (αMSH) secretion from the hypothalamus, elevated blood pressure and heart rate (HR), increased brown adipose tissue (BAT) thermogenesis, and reduced both food intake (FI) and body weight (BW). In melanocortin 4 receptor-deficient (MC4R knockout [KO]) mice, metabolic and cardiovascular effects of AMPH were significantly attenuated. Antagonism of serotonergic and noradrenergic neurotransmitter systems attenuated AMPH-induced αMSH secretion as well as AMPH-induced metabolic and cardiovascular effects. We propose that AMPH increases serotonergic activation of proopiomelanocortin (POMC) neurons and reduces the noradrenergic inhibition of POMC neurons, thereby disinhibiting them. Together, these presynaptic mechanisms result in increased POMC activity, increased αMSH secretion, and increased activation of MC4R pathways that regulate both the metabolic and cardiovascular systems. Show less

Historically, 17β-hydroxysteroid dehydrogenase type 3 (HSD17B3) was thought to be the key enzyme responsible for testicular testosterone production. In humans, loss-of-function mutations in HSD17B3 impair testosterone production during prenatal life leading to impaired development of androgen-dependent tissues in 46,XY individuals. However, male mice with HSD17B3 deficiency exhibit normal testicular testosterone concentrations, normal development of reproductive organs and are fertile, suggesting that mice express other hydroxysteroid dehydrogenase enzymes capable of testicular testosterone synthesis. This study aimed to investigate whether 17β-hydroxysteroid dehydrogenase type 12 (HSD17B12), which can convert androstenedione to testosterone in mice but not in humans, compensates for the lack of HSD17B3 in Hsd17b3 knockout (KO) mice. We used CRISPR/Cas9 to substitute the amino acid in mouse HSD17B12 that is responsible for its ability to convert androstenedione to testosterone with the amino acid of the human enzyme that prevents androstenedione being used as a substrate. When this Hsd17b12 mutation was introduced into Hsd17b3 KO mice, males exhibited normal reproductive tracts but reduced testicular testosterone production with a consequential reduction in seminal vesicle weight. This suggests HSD17B12 contributes toward testosterone production in the absence of HSD17B3, but other enzymes must also contribute. We therefore quantified other testicular hydroxysteroid dehydrogenases, finding that HSD17B7 mRNA and protein was markedly upregulated in Hsd17b3 KO testes. We confirmed that mouse, but not human, HSD17B7 can produce testosterone in vitro. We conclude that compared to humans, mice exhibit increased plasticity in testosterone production via hydroxysteroid dehydrogenase enzymes to support androgen action and male fertility. Show less

Cannabis is widely used worldwide, yet its links to health outcomes are not fully understood. DNA methylation can serve as a mediator to link environmental exposures to health outcomes. We conducted an epigenome-wide association study (EWAS) of peripheral blood-based DNA methylation and lifetime cannabis use (ever vs. never) in a meta-analysis including 9436 participants (7795 European and 1641 African ancestry) from seven cohorts. Accounting for effects of cigarette smoking, our trans-ancestry EWAS meta-analysis revealed four CpG sites significantly associated with lifetime cannabis use at a false discovery rate of 0.05 Show less

Testosterone and dihydrotestosterone (DHT) are essential for male development and fertility. In the canonical androgen production pathway, testosterone is produced in the testis by HSD17B3; however, adult male Hsd17b3 knockout (KO) mice continue to produce androgens and are fertile, indicating compensatory mechanisms exist. A second, alternate pathway produces DHT from precursors other than testosterone via 5α-reductase (SRD5A) activity. We hypothesized that the alternate pathway contributes to androgen bioactivity in Hsd17b3 KO mice. To investigate contributions arising from and interactions between the canonical and alternate pathways, we pharmacologically inhibited SRD5A and ablated Srd5a1 (the predominant SRD5A in the testis) on the background of Hsd17b3 KO mice. Mice with perturbation of either the canonical or both pathways exhibited increased LH, testicular steroidogenic enzyme expression, and normal reproductive tracts and fertility. In the circulation, alternate pathway steroids were increased in the absence of HSD17B3 but were reduced by co-inhibition of SRD5A1. Mice with perturbations of both pathways produced normal basal levels of intratesticular testosterone, suggesting the action of other unidentified hydroxysteroid dehydrogenase(s). Strikingly, testicular expression of another SRD5A enzyme, Srd5a2, was markedly increased in the absence of Hsd17b3, suggesting a compensatory increase in SRD5A2 to maintain androgen bioactivity during HSD17B3 deficiency. Finally, we observed elevated circulating concentrations of the 11-keto-derivative of DHT, suggesting compensatory extra-gonadal induction of bioactive 11-keto androgen production. Taken together, we conclude that, in the absence of the canonical pathway of androgen production, multiple intra- and extra-gonadal mechanisms cooperate to maintain testosterone and DHT production, supporting male development and fertility. Show less

Androgens such as testosterone and dihydrotestosterone (DHT) are essential for male sexual development, masculinisation, and fertility. Testosterone is produced via the canonical androgen production pathway and is essential for normal masculinisation and testis function. Disruption to androgen production can result in disorders of sexual development (DSD). In the canonical pathway, 17β-hydroxysteroid dehydrogenase type 3 (HSD17B3) is viewed as a critical enzyme in the production of testosterone, performing the final conversion required. HSD17B3 deficiency in humans is associated with DSD due to low testosterone concentration during development. Individuals with Show less

The only known non-pharmacological means to alter long chain polyunsaturated fatty acid (LCPUFA) abundance in mammalian tissue is by altering substrate fatty acid ratios. Alternative mRNA splicing is increasingly recognized as a modulator of protein structure and function. Here we report identification of a novel alternative transcript (AT) of fatty acid desaturase 2 (FADS2) that inhibits production of omega-3 but not omega-6 LCPUFA, discovered during study of ATs in human milk fat globules (MFG). Human breastmilk collected from a single donor was used to isolate MFG. An mRNA-sequencing library was constructed from the total RNA isolated from the MFG. The constructed library was sequenced using an Illumina HiSeq instrument operating in high output mode. Expression levels of evolutionary conserved FADSAT were measured using cDNA from MFG by semi-quantitative RT-PCR assay. RNA sequencing revealed >15,000 transcripts, including moderate expression of the FADS2 classical transcript (CS). A novel FADS2 alternative transcript (FADS2AT2) with 386 amino acids was discovered. When FADS2AT2 was transiently transfected into MCF7 cells stably expressing FADS2, delta-6 desaturation (D6D) of alpha-linolenic acid 18:3n-3 → 18:4n-3 was suppressed as were downstream products 20:4n-3 and 20:5n-3. In contrast, no significant effect on D6D of linoleic acid 18:2n-6 → 18:3n-6 or downstream products was observed. FADS2, FADS2AT1 and 5 out of 8 known FADS3AT were expressed in MFG. FADS1, FADS3AT3, and FADS3AT5 are undetectable. The novel, noncatalytic FADS2AT2 regulates FADS2CS-mediated Δ6-desaturation of omega-3 but not omega-6 PUFA biosynthesis. This spliced isoform mediated interaction is the first molecular mechanism by which desaturation of one PUFA family but not the other is modulated. Show less

Numerous genetic alterations of HSA 11q13 are found frequently in several cancer types, including breast cancer (BC). The 11q13 locus harbors FADS2 encoding Δ6 desaturation which is not functional in several cancer cell lines, including hormone positive MCF7 BC cells. In vitro, the non-functional FADS2 activity unmasks 18:2n-6 elongation to 20:2n-6 and Δ5 desaturation by FADS1 to yield 5Z,11Z,14Z-20:3 (sciadonic acid) rather than 5Z,8Z,11Z,14Z-20:4 (arachidonic acid). In this pilot study we aimed to determine whether 5,11,14-20:3 appears in vivo in hormone positive human BC tissue. Fatty acids were profiled in surgically removed human breast tumor and adjacent normal tissue (n = 9). Sciadonic acid was detected in three of nine breast tumor samples and was below detect limits in normal breast tissue. The internal Δ8 double bond of arachidonic acid is required for normal eicosanoid synthesis but is missing in sciadonic acid. This pilot study demonstrates for the first time in vivo sciadonic acid in hormone positive BC tissue, warranting a larger survey study to further evaluate its appearance and the functional implications. Show less

In mammals, FADS2 catalyzes "front-end" Δ4-, Δ6-, and Δ8-desaturation of fatty acyl chains, whereas FADS1 has Δ5-desaturase activity. Eighteen and 20-carbon precursors to highly unsaturated n-3 and n-6 fatty acids are the usual substrates for FADS1 and FADS2. Our main objective was to characterize the metabolic fate of oleic acid (OA) due to action of FADS gene products. MCF-7 cells were stably transformed with either FADS1 or FADS2 or empty vector. A series of dose-response experiments were conducted with albumin-bound fatty acid substrates (18:1n-9 and 20:1n-9) provided in concentrations up to 100µM. Cells were harvested after 24h, after which FAME were prepared and analyzed by GC-FID and covalent adduct chemical ionization tandem mass spectrometry (CACI-MS/MS). When stably transformed cells were incubated with 18:1n-9, FADS1 and control cells elongated 18:1n-9 → 20:1n-9 (11-20:1), while FADS2 cells Δ6 desaturated, elongated, and then Δ5 desaturated via FADS1 coded activity leading to Mead acid, 9-18:1 → 6,9-18:2 → 8,11-20:2 (20:2n-9) → 6,8,11-20:3 (20:3n-9). Surprisingly, FADS1 cells Δ7 desaturated 11-20:1 → 7,11-20:2, the latter detected at low levels in control and FADS2 cells. Our results imply three pathways operate on 18:1n-9: 1) 18:1n-9 → 18:2n-9 → 20:2n-9 → 20:3n-9; 2) 18:1n-9 → 20:1n-9 → 20:2n-9 → 20:3n-9 and 3) 18:1n-9 → 20:1n-9 → 7,11-20:2. Alternative pathways for oleic acid metabolism exist depending on FADS2 or FADS1 activities, we present the first evidence of Δ7 desaturation via the FADS1 gene product. Show less

Fatty acid desaturase 3 (FADS3) is the third member of the FADS gene cluster. FADS1 and FADS2 code for enzymes required for highly unsaturated fatty acid (HUFA) biosynthesis, but FADS3 function remains elusive. We generated the first Fads3 knockout (KO) mouse with an aim to characterize its metabolic phenotype and clues to in vivo function. All mice (wild type (WT) and KO) were fed facility rodent chow devoid of HUFA. No differences in overt phenotypes (survival, fertility, growth rate) were observed. Docosahexaenoic acid (DHA, 22:6n-3) levels in the brain of postnatal day 1 (P1) KO mice were lower than the WT (P < 0.05). The ratio of docosapentaenoic acid (DPA, 22:5n-3) to DHA in P1 KO liver was higher than in WT suggesting lower desaturase activity. Concomitantly, 20:4n-6 was lower but its elongation product 22:4n-6 was greater in the liver of P1 KO mice. P1 KO liver Fads1 and Fads2 mRNA levels were significantly downregulated whereas expression levels of elongation of very long chain 2 (Elovl2) and Elovl5 genes were upregulated compared to age-matched WT. No Δ13-desaturation of vaccenic acid was observed in liver or heart in WT mice expressing FADS3 as was reported in vitro. Taken together, the fatty acid compositional results suggest that Fads3 enhances liver-mediated 22:6n-3 synthesis to support brain 22:6n-3 accretion before and during the brain growth spurt. Show less

Adeno-associated virus 2 (AAV2) and adenovirus 5 (Ad5) are promising gene therapy vectors. Both display liver tropism and are currently thought to enter hepatocytes in vivo through cell surface heparan sulfate proteoglycans (HSPGs). To test directly this hypothesis, we created mice that lack Ext1, an enzyme required for heparan sulfate biosynthesis, in hepatocytes. Ext1(HEP) mutant mice exhibit an 8-fold reduction of heparan sulfate in primary hepatocytes and a 5-fold reduction of heparan sulfate in whole liver tissue. Conditional hepatocyte Ext1 gene deletion greatly reduced AAV2 liver transduction following intravenous injection. Ad5 transduction requires blood coagulation factor X (FX); FX binds to the Ad5 capsid hexon protein and bridges the virus to HSPGs on the cell surface. Ad5.FX transduction was abrogated in primary hepatocytes from Ext1(HEP) mice. However, in contrast to the case with AAV2, Ad5 transduction was not significantly reduced in the livers of Ext1(HEP) mice. FX remained essential for Ad5 transduction in vivo in Ext1(HEP) mice. We conclude that while AAV2 requires HSPGs for entry into mouse hepatocytes, HSPGs are dispensable for Ad5 hepatocyte transduction in vivo. This study reopens the question of how adenovirus enters cells in vivo. Our understanding of how viruses enter cells, and how they can be used as therapeutic vectors to manage disease, begins with identification of the cell surface receptors to which viruses bind and which mediate viral entry. Both adeno-associated virus 2 and adenovirus 5 are currently thought to enter hepatocytes in vivo through heparan sulfate proteoglycans (HSPGs). However, direct evidence for these conclusions is lacking. Experiments presented herein, in which hepatic heparan sulfate synthesis was genetically abolished, demonstrated that HSPGs are not likely to function as hepatocyte Ad5 receptors in vivo. The data also demonstrate that HSPGs are required for hepatocyte transduction by AAV2. These results reopen the question of the identity of the Ad5 receptor in vivo and emphasize the necessity of demonstrating the nature of the receptor by genetic means, both for understanding Ad5 entry into cells in vivo and for optimization of Ad5 vectors as therapeutic agents. Show less

Female humans and rodents have been shown to have higher 22:6n-3 status and synthesis than males. It is unclear which sex hormone is involved. We investigated the specificity of the effects of physiological concentrations of sex hormones in vitro on the mRNA expression of genes involved in polyunsaturated fatty acid (PUFA) biosynthesis and on the conversion of [d5]-18:3n-3 to longer chain fatty acids. Progesterone, but not 17α-ethynylestradiol or testosterone, increased FADS2, FADS1, ELOVl 5 and ELOVl 2 mRNA expression in HepG2 cells, but only FADS2 in primary human hepatocytes. In HepG2 cells, these changes were accompanied by hypomethylation of specific CpG loci in the FADS2 promoter. Progesterone, not 17α-ethynylestradiol or testosterone, increased conversion of [d5]-18:3n-3 to 20:5n-3, 22:5n-3 and 22:6n-3. These findings show that progesterone increases n-3 PUFA biosynthesis by up-regulating the mRNA expression of genes involved in this pathway, possibly via changes in the epigenetic regulation of FADS2. Show less

Fungi are the second most abundant type of human pathogens. Invasive fungal pathogens are leading causes of life-threatening infections in clinical settings. Toxicity to the host and drug-resistance are two major deleterious issues associated with existing antifungal agents. Increasing a host's tolerance and/or immunity to fungal pathogens has potential to alleviate these problems. A host's tolerance may be improved by modulating the immune system such that it responds more rapidly and robustly in all facets, ranging from the recognition of pathogens to their clearance from the host. An understanding of biological processes and genes that are perturbed during attempted fungal exposure, colonization, and/or invasion will help guide the identification of endogenous immunomodulators and/or small molecules that activate host-immune responses such as specialized adjuvants. In this study, we present computational techniques and approaches using publicly available transcriptional data sets, to predict immunomodulators that may act against multiple fungal pathogens. Our study analyzed data sets derived from host cells exposed to five fungal pathogens, namely, Alternaria alternata, Aspergillus fumigatus, Candida albicans, Pneumocystis jirovecii, and Stachybotrys chartarum. We observed statistically significant associations between host responses to A. fumigatus and C. albicans. Our analysis identified biological processes that were consistently perturbed by these two pathogens. These processes contained both immune response-inducing genes such as MALT1, SERPINE1, ICAM1, and IL8, and immune response-repressing genes such as DUSP8, DUSP6, and SPRED2. We hypothesize that these genes belong to a pool of common immunomodulators that can potentially be activated or suppressed (agonized or antagonized) in order to render the host more tolerant to infections caused by A. fumigatus and C. albicans. Our computational approaches and methodologies described here can now be applied to newly generated or expanded data sets for further elucidation of additional drug targets. Moreover, identified immunomodulators may be used to generate experimentally testable hypotheses that could help in the discovery of broad-spectrum immunotherapeutic interventions. All of our results are available at the following supplementary website: http://bioinformatics.cs.vt.edu/~murali/supplements/2013-kidane-bmc. Show less

Itraconazole (ITZ) is an approved antifungal agent that carries a "black box warning" in its label regarding a risk of negative cardiac inotropy based on clinical findings. Since the mechanism of the negative inotropic effect is unknown, we performed a variety of preclinical and mechanistic studies to explore the pharmacological profile of ITZ and understand the negative inotropic mechanism. ITZ was evaluated in: (1) an isolated rabbit heart (IRH) preparation using Langendorff retrograde perfusion; (2) ion channel studies; (3) a rat heart mitochondrial function profiling screen; (4) a mitochondrial membrane potential (MMP) assay; (5) in vitro pharmacology profiling assays (148 receptors, ion channels, transporters, and enzymes); and (6) a kinase selectivity panel (451 kinases). In the IRH, ITZ decreased cardiac contractility (>30%) at 0.3μM, with increasing effect at higher concentrations, which indicated a direct negative inotropic effect upon the heart. It also decreased heart rate and coronary flow (≥1μM) and prolonged PR/QRS intervals (3μM). In mechanistic studies, ITZ inhibited the cardiac NaV channel (IC50: 4.2μM) and was devoid of any functional inhibitory effect at the remaining pharmacological targets. Lastly, ITZ did not affect MMP, nor interfere with mitochondrial enzymes or processes involved with fuel substrate utilization or energy formation. Overall, the cardiovascular and mechanistic data suggest that ITZ-induced negative inotropy is a direct effect on the heart, in addition, the potential involvement of mitochondria function and L-type Ca(2+) channels are eliminated. The exact mechanism underlying the negative inotropy is uncertain, and requires further study. Show less

Inherited defects in the ability to catabolize glycosaminoglycans result in lysosomal storage disorders known as mucopolysaccharidoses (MPS), causing severe pathology, particularly in the brain. Enzyme replacement therapy has been used to treat mucopolysaccharidoses; however, neuropathology has remained refractory to this approach. To test directly whether substrate reduction might be feasible for treating MPS disease, we developed a genetic model for substrate reduction therapy by crossing MPS IIIa mice with animals partially deficient in heparan sulfate biosynthesis due to heterozygosity in Ext1 and Ext2, genes that encode the copolymerase required for heparan sulfate chain assembly. Reduction of heparan sulfate by 30-50% using this genetic strategy ameliorated the amount of disease-specific biomarker and pathology in multiple tissues, including the brain. In addition, we were able to demonstrate that substrate reduction therapy can improve the efficacy of enzyme replacement therapy in cell culture and in mice. These results provide proof of principle that targeted inhibition of heparan sulfate biosynthetic enzymes together with enzyme replacement might prove beneficial for treating mucopolysaccharidoses. Show less

The fatty acid desaturase (FADS) genes code for the rate-limiting enzymes required for the biosynthesis of long-chain polyunsaturated fatty acids (LCPUFA). Here we report discovery and function of a novel FADS1 splice variant. FADS1 alternative transcript 1 (FADS1AT1) enhances desaturation of FADS2, leading to increased production of eicosanoid precursors, the first case of an isoform modulating the enzymatic activity encoded by another gene. Multiple protein isoforms were detected in primate liver, thymus, and brain. In human neuronal cells, their expression patterns are modulated by differentiation and result in alteration of cellular fatty acids. FADS1, but not FADS1AT1, localizes to endoplasmic reticulum and mitochondria. Ribosomal footprinting demonstrates that all three FADS genes are translated at similar levels. The noncatalytic regulation of FADS2 desaturation by FADS1AT1 is a novel, plausible mechanism by which several phylogenetically conserved FADS isoforms may regulate LCPUFA biosynthesis in a manner specific to tissue, organelle, and developmental stage. Show less

Genes coding for the fatty acid desaturases (FADS1, 2, 3) localized at the cancer genomic hotspot 11q13 locus are required for the biosynthesis of 20 carbon polyunsaturated fatty acids (PUFA) that are direct eicosanoid precursors. In several cancer cell lines, FADS2 encoded Δ6 and Δ8 desaturation is not functional. Analyzing MCF7 cell fatty acids with detailed structural mass spectrometry, we show that in the absence of FADS2 activity, the FADS1 product Δ5-desaturase operates to produce 5,11,14-20∶3 and 5,11,14,17-20∶4. These PUFA are missing the 8-9 double bond of the eicosanoid signaling precursors arachidonic acid (5,8,11,14-20∶4) and eicosapentaenoic acid (5,8,11,14,17-20∶5). Heterologous expression of FADS2 restores Δ6 and Δ8-desaturase activity and normal eicosanoid precursor synthesis. The loss of FADS2-encoded activities in cancer cells shuts down normal PUFA biosynthesis, deleting the endogenous supply of eicosanoid and downstream docosanoid precursors, and replacing them with unusual butylene-interrupted fatty acids. If recapitulated in vivo, the normal eicosanoid and docosanoid cell signaling milieu would be depleted and altered due to reduction and substitution of normal substrates with unusual substrates, with unpredictable consequences for cellular communication. Show less

The Δ6 desaturase, encoded by FADS2, plays a crucial role in omega-3 and omega-6 fatty acid synthesis. These fatty acids are essential components of the central nervous system, and they act as precursors for eicosanoid signaling molecules and as direct modulators of gene expression. The polypyrimidine tract binding protein (PTB or hnRNP I) is a splicing factor that regulates alternative pre-mRNA splicing. Here, PTB is shown to bind an exonic splicing silencer element and repress alternative splicing of FADS2 into FADS2 AT1. PTB and FADS2AT1 were inversely correlated in neonatal baboon tissues, implicating PTB as a major regulator of tissue-specific FADS2 splicing. In HepG2 cells, PTB knockdown modulated alternative splicing of FADS2, as well as FADS3, a putative desaturase of unknown function. Omega-3 fatty acids decreased by nearly one half relative to omega-6 fatty acids in PTB knockdown cells compared with controls, with a particularly strong decrease in eicosapentaenoic acid (EPA) concentration and its ratio to arachidonic acid (ARA). This is a rare demonstration of a mechanism specifically altering the cellular omega-3 to omega-6 fatty acid ratio without any change in diet/media. These findings reveal a novel role for PTB, regulating availability of membrane components and eicosanoid precursors for cell signaling. Show less

Obesity is globally prevalent and highly heritable, but its underlying genetic factors remain largely elusive. To identify genetic loci for obesity susceptibility, we examined associations between body mass index and ∼ 2.8 million SNPs in up to 123,865 individuals with targeted follow up of 42 SNPs in up to 125,931 additional individuals. We confirmed 14 known obesity susceptibility loci and identified 18 new loci associated with body mass index (P < 5 × 10⁻⁸), one of which includes a copy number variant near GPRC5B. Some loci (at MC4R, POMC, SH2B1 and BDNF) map near key hypothalamic regulators of energy balance, and one of these loci is near GIPR, an incretin receptor. Furthermore, genes in other newly associated loci may provide new insights into human body weight regulation. Show less

Markers at the pericentriolar material 1 gene (PCM1) have shown genetic association with schizophrenia in both a University College London (UCL) and a USA-based case-control sample. In this paper we report a statistically significant replication of the PCM1 association in a large Scottish case-control sample from Aberdeen. Resequencing of the genomic DNA from research volunteers who had inherited haplotypes associated with schizophrenia showed a threonine to isoleucine missense mutation in exon 24 which was likely to change the structure and function of PCM1 (rs370429). This mutation was found only as a heterozygote in 98 schizophrenic research subjects and controls out of 2246 case and control research subjects. Among the 98 carriers of rs370429, 67 were affected with schizophrenia. The same alleles and haplotypes were associated with schizophrenia in both the London and Aberdeen samples. Another potential aetiological base pair change in PCM1 was rs445422, which altered a splice site signal. A further mutation, rs208747, was shown by electrophoretic mobility shift assays to create or destroy a promoter transcription factor site. Five further non-synonymous changes in exons were also found. Genotyping of the new variants discovered in the UCL case-control sample strengthened the evidence for allelic and haplotypic association (P=0.02-0.0002). Given the number and identity of the haplotypes associated with schizophrenia, further aetiological base pair changes must exist within and around the PCM1 gene. PCM1 protein has been shown to interact directly with the disrupted-in-schizophrenia 1 (DISC1) protein, Bardet-Biedl syndrome 4, and Huntingtin-associated protein 1, and is important in neuronal cell growth. In a separate study we found that clozapine but not haloperidol downregulated PCM1 expression in the mouse brain. We hypothesize that mutant PCM1 may be responsible for causing a subtype of schizophrenia through abnormal cell division and abnormal regeneration in dividing cells in the central nervous system. This is supported by our previous finding of orbitofrontal volumetric deficits in PCM1-associated schizophrenia patients as opposed to temporal pole deficits in non-PCM1-associated schizophrenia patients. Caution needs to be exercised in interpreting the actual biological effects of the mutations we have found without further cell biology. However, the DNA changes we have found deserve widespread genotyping in multiple case-control populations. Show less

Recent studies have reported large common regions of homozygosity (ROHs) that are the result of autozygosity, that is, the cooccurrence within individuals of long haplotypes that have a high frequency in the population. A recent study reports that such regions are found more commonly in individuals with schizophrenia compared with controls, and identified nine 'risk ROHs' that were individually more common in cases. Of these, four contained or neighboured genes associated with schizophrenia (NOS1AP/UHMK1, ATF2, NSF and PIK3C3). We have applied the same methodology to a UK sample of 506 cases with bipolar disorder and 510 controls. There was no overall excess of common ROHs among bipolar individuals. With one exception, the haplotypes accounting for the ROHs appeared to be distributed according to the Hardy-Weinberg equilibrium. One ROH was individually more common among cases (uncorrected P = 0.0003). This ROH spanned the chromosome 2p23.3 gene ITSN2 (the gene for intersectin 2 isoform 2). However, inspection of the homozygous haplotypes and haplotype-based tests for association failed to provide a clearer understanding of why this ROH was occurring more commonly. Overall, we conclude that, in contrast with schizophrenia, common ROHs are rarely associated with susceptibility to bipolar disorder. This supports the idea that predominantly different genes are increasing susceptibility to schizophrenia and bipolar affective disorders. Show less

Oestradiol (E2) stimulates the growth of hormone-dependent breast cancer. 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyse the pre-receptor activation/inactivation of hormones and other substrates. 17beta-HSD1 converts oestrone (E1) to active E2, but it has recently been suggested that another 17beta-HSD, 17beta-HSD12, may be the major enzyme that catalyses this reaction in women. Here we demonstrate that it is 17beta-HSD1 which is important for E2 production and report the inhibition of E1-stimulated breast tumor growth by STX1040, a non-oestrogenic selective inhibitor of 17beta-HSD1, using a novel murine model. 17beta-HSD1 and 17beta-HSD12 mRNA and protein expression, and E2 production, were assayed in wild type breast cancer cell lines and in cells after siRNA and cDNA transfection. Although 17beta-HSD12 was highly expressed in breast cancer cell lines, only 17beta-HSD1 efficiently catalysed E2 formation. The effect of STX1040 on the proliferation of E1-stimulated T47D breast cancer cells was determined in vitro and in vivo. Cells inoculated into ovariectomised nude mice were stimulated using 0.05 or 0.1 microg E1 (s.c.) daily, and on day 35 the mice were dosed additionally with 20 mg/kg STX1040 s.c. daily for 28 days. STX1040 inhibited E1-stimulated proliferation of T47D cells in vitro and significantly decreased tumor volumes and plasma E2 levels in vivo. In conclusion, a model was developed to study the inhibition of the major oestrogenic 17beta-HSD, 17beta-HSD1, in breast cancer. Both E2 production and tumor growth were inhibited by STX1040, suggesting that 17beta-HSD1 inhibitors such as STX1040 may provide a novel treatment for hormone-dependent breast cancer. Show less

Endocrine therapy of prostate cancer (PCa) relies on agents which disrupt the biosynthesis of testosterone in the testis and/or by direct antagonism of active hormone on the androgen receptor (AR) in non-gonadal target tissues of hormone action such as the prostate. In an effort to evaluate new therapies which could inhibit gonadal or non-gonadal testosterone biosynthesis, we developed high throughput biochemical and cellular screening assays to identify inhibitors of 17beta-hydroxysteroid dehydrogenase type III (17beta-HSD3), the enzyme catalyzing the conversion of androstenedione (AdT) to testosterone. Initial screening efforts identified a natural product, 18beta-glycyrrhetinic acid, and a novel derivative of AdT, 3-O-benzylandrosterone, as potent inhibitors of the enzyme. Further efforts led to the identification of several classes of non-steroidal, low molecular weight compounds that potently inhibited 17beta-HSD3 enzymatic activity. One of the most potent classes of 17beta-HSD3 inhibitors was a series of anthranilamide small molecules identified from a collection of compounds related to non-steroidal modulators of nuclear hormone receptors. The anthranilamide based 17beta-HSD3 inhibitors were exemplified by BMS-856, a compound displaying low nanomolar inhibition of 17beta-HSD3 enzymatic activity. In addition, this series of compounds displayed potent inhibition of 17beta-HSD3-mediated cellular conversion of AdT to testosterone and inhibited the 17beta-HSD3-mediated conversion of testosterone necessary to promote AR-dependent transcription. The identification of non-steroidal functional inhibitors of 17beta-HSD3 may be a useful complementary approach for the disruption of testosterone biosynthesis in the treatment of PCa. Show less