Search

Much is known about growth and nutrient uptake traits and ecological stoichiometry in natural systems. However, these concepts have been comparatively understudied in agricultural systems despite their potential to infer nutrient limitation and interspecific resource competition. This study established a model mixed pasture system to assess tissue C:N and C:P stoichiometry and aboveground biomass (AGB) in a grass (Phalaris aquatica) and legume (Trifolium vesiculosum) under factorial inputs of high and low nitrogen (N) and phosphorus (P), in monoculture and mixture. Due to inherent trait diversity, we expected grass and legume growth, shoot vs root stoichiometry and N:P homeostasis to differ in response to nutrient limitation and between monoculture and mixture. Grass AGB was greater with N addition and in mixture, and legume AGB was decreased by N but increased by P, more so in mixture. Nutrient limitation in grass was determined via a strong coupling of growth with shoot stoichiometry, by which AGB decreased and C:N increased under N limitation. Legume growth was not correlated with tissue stoichiometry, but potential for growth limitation by N and P was detected via increased shoot C:N under low N and P, and C:P under low P. Legume shoot N:P was more homeostatic than grass, and grass shoot N:P homeostasis was greater in mixtures than in monocultures. Integrating ecological stoichiometry alongside trait-based ecology is a useful tool for predicting how fertiliser management may affect nutrient balance and species dominance in mixed pasture agroecosystems.

BACKGROUND Prenatal exposure to alcohol leads to a greater incidence of many cardiovascular-related diseases, presumably via a mechanism that may involve increased oxidative stress. An agonist of peroxisome proliferator-activated receptor gamma (PPARγ; rosiglitazone) has been shown to suppress alcohol-induced neuroinflammation and oxidative stress. The goal of this study was to determine whether acute and chronic treatment with rosiglitazone could restore or prevent impaired nitric oxide synthase (NOS)-dependent responses of cerebral arterioles in male and female adult (14-16 weeks old) rats exposed to alcohol in utero. METHODS We fed Sprague-Dawley dams a liquid diet with or without 3% ethanol for the duration of their pregnancy (21-23 days). In the first series of studies, we examined the reactivity of cerebral arterioles to eNOS- (ADP), nNOS-dependent (NMDA), and NOS-independent agonists in male and female adult rats before and during acute (1 hour) topical application of rosiglitazone (1 µM). In a second series of studies, we examined the influence of chronic treatment with rosiglitazone (3 mg/kg/day in drinking water for 2-3 weeks) on the responses of cerebral arterioles in male and female adult rats exposed to alcohol in utero. RESULTS We found that in utero exposure to alcohol similarly reduced responses of cerebral arterioles to ADP and NMDA, but not to nitroglycerin in male and female adult rats. In addition, acute treatment of the male and female adult rats with rosiglitazone similarly restored this impairment in cerebral vascular function to that observed in controls. We also found that chronic treatment with rosiglitazone prevented impaired vascular function in male and female adult rats that were exposed to alcohol in utero. CONCLUSIONS PPARγ activation may be an effective and relevant treatment to reverse or prevent cerebral vascular abnormalities associated with prenatal exposure to alcohol.

Emerged from the German Association for Cannabis as Medicine (ACM), a handful of enthusiastic medical doctors from Germany, Switzerland, and Austria supported by experts from other countries established the International Association for Cannabinoid Medicines (IACM) in March 2000. During the past 20 years until today, it evolved toward the most important scientific society for clinical research in the field of cannabis-based medicines. The overarching aim of the IACM is to advance the knowledge on cannabis, cannabinoids, the endocannabinoid system, and related topics especially with regard to their therapeutic potential, which would allow more patients to profit from a treatment with cannabis-based medicines and ease legislators to adopt their policies. To reach this goal, the IACM organizes international scientific conferences, biweekly publishes the IACM-Bulletin—currently in six different languages—reaching more than 5000 people worldwide, and is working closely together with patient associations, international partner organizations, and IACM ambassadors. In 2019, a collaborative partnership with the journal Cannabis and Cannabinoid Research was established.

Efficient isolation of genetically modified cells that are phenotypically indistinguishable from the unmodified cells remains a major technical barrier for the broader utilization of CRISPR/Cas9. Here, we report a novel enrichment approach to select the genome engineered cells by co-targeting a genomically integrated GFP gene along with the endogenous gene of interest (GOI). Using this co-targeting approach, multiple genomic loci were successfully targeted in chicken (DF1) and quail (CEC-32) fibroblast cell lines by transient transfection of Cas9 and guide RNAs (gRNAs). Clonal isolation of co-targeted DF1 cells showed 75% of cell clones had deletion of GFP and biallelic deletion of the GOI. To assess the utility of this approach to generate genome modified animals, we tested it on chicken primordial germ cells (PGCs) expressing GFP by co-targeting with gRNAs against GFP and endogenous ovomucoid (OVM) gene. PGCs enriched for loss of GFP and confirmed for OVM deletion, derived by co-targeting, were injected into Hamburger and Hamilton stage 14-15 chicken embryos, and their ability to migrate to the genital ridge was confirmed. This simple, efficient enrichment approach could easily be applied to the creation of knock-out or edited cell lines or animals.

Serine synthase (SS) from Fusobacterium nucleatum is a fold type II pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the β-replacement of l-cysteine with water to form l-serine and H2S. Herein, we show that SS can also function as a cysteine synthase, catalyzing the β-replacement of l-serine with bisulfide to produce l-cysteine and H2O. The forward (serine synthase) and reverse (cysteine synthase) reactions occur with comparable turnover numbers and catalytic efficiencies for the amino acid substrate. Reaction of SS with l-cysteine leads to transient formation of a quinonoid species, suggesting that deprotonation of the Cα and β-elimination of the thiolate group from l-cysteine occur via a stepwise mechanism. In contrast, the quinonoid species was not detected in the formation of the α-aminoacrylate intermediate following reaction of SS with l-serine. A key active site residue, D232, was shown to stabilize the more chemically reactive ketoenamine PLP tautomer and also function as an acid/base catalyst in the forward and reverse reactions. Fluorescence resonance energy transfer between PLP and W99, the enzyme's only tryptophan residue, supports ligand-induced closure of the active site, which shields the PLP cofactor from the solvent and increases the basicity of D232. These results provide new insight into amino acid metabolism in F. nucleatum and highlight the multiple catalytic roles of D232 in a new member of the fold type II family of PLP-dependent enzymes.

Resistant tumours are thought to arise from the action of Darwinian selection on genetically heterogenous cancer cell populations. However, simple clonal selection is inadequate to describe the late relapses often characterising luminal breast cancers treated with endocrine therapy (ET), suggesting a more complex interplay between genetic and non-genetic factors. Here, we dissect the contributions of clonal genetic diversity and transcriptional plasticity during the early and late phases of ET at single-cell resolution. Using single-cell RNA-sequencing and imaging we disentangle the transcriptional variability of plastic cells and define a rare subpopulation of pre-adapted (PA) cells which undergoes further transcriptomic reprogramming and copy number changes to acquire full resistance. We find evidence for sub-clonal expression of a PA signature in primary tumours and for dominant expression in clustered circulating tumour cells. We propose a multi-step model for ET resistance development and advocate the use of stage-specific biomarkers., The development of resistance to endocrine therapy is a significant, clinical problem in breast cancer. Here, the authors identify a rare subpopulation of cells that drive resistance following transcriptional reprogramming.

Lanthionine synthase from the oral bacterium Fusobacterium nucleatum is a fold type II pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes the β-replacement of l-cysteine by a second molecule of l-cysteine to form H2S and l-lanthionine. The meso-isomer of the latter product is incorporated into the F. nucleatum peptidoglycan layer. Herein, we investigated the catalytic role of S224, which engages in hydrogen-bond contact with the terminal carboxylate of l-lanthionine in the closed conformation of the enzyme. Unexpectedly, the S224A variant elicited a 7-fold increase in the turnover rate for H2S and lanthionine formation and a 70-fold faster rate constant for the formation of the α-aminoacrylate intermediate compared to the wild-type enzyme. Presteady state kinetic analysis further showed that the reaction between S224A and l-cysteine leads to the formation of the more reactive ketoenamine tautomer of the α-aminoacrylate. The α-aminoacrylate with the protonated Schiff base is not an observable intermediate in the analogous reaction with the wild type, which may account for its attenuated kinetic properties. However, the S224A substitution is detrimental to other aspects of the catalytic cycle; it facilitates the α,β-elimination of l-lanthionine, and it weakens the enzyme's catalytic preference for the formation of l-lanthionine over that of l-cystathionine.

Development of simple and readily adoptable methods to mediate germline engineering of the chicken genome will have many applications in research, agriculture and industrial biotechnology. We report germline targeting of the endogenous chicken Interferon Alpha and Beta Receptor Subunit 1 (IFNAR1) gene by in vivo transgenic expression of the high-fidelity Cas9 (Cas9-HF1) and guide RNAs (gRNAs) in chickens. First, we developed a Tol2 transposon vector carrying Cas9-HF1, IFNAR1-gRNAs (IF-gRNAs) and green fluorescent protein (GFP) transgenes (pTgRCG) and validated in chicken fibroblast DF1 cells. Next, the pTgRCG plasmid was directly injected into the dorsal aorta of embryonic day (ED) 2.5 chicken embryos targeting the circulating primordial germ cells (PGCs). The resulting chimera roosters generated a fully transgenic generation 1 (G1) hen with constitutive expression of Cas9-HF1 and IF-gRNAs (G1_Tol2-Cas9/IF-gRNA). We detected a spectrum of indels at gRNA-targeted loci in the G1_Tol2-Cas9/IF-gRNA hen and the indels were stably inherited by the G2 progeny. Breeding of the G1_Tol2-Cas9/IF-gRNA hen resulted in up to 10% transgene-free heterozygote IFNAR1 mutants, following null-segregation of the Tol2 insert. The method described here will provide new opportunities for genome editing in chicken and other avian species that lack PGC culture.

ZUSAMMENFASSUNGDie Behandlung von Patienten mit Gilles-de-la-Tourette-Syndrom (GTS) ist bis heute unbefriedigend, da etablierte Therapien nur zu einer partiellen Reduktion der Tics und nicht zu einer Verbesserung psychiatrischer Komorbiditäten führen. Die medikamentöse Behandlung mit Antipsychotika führt oft zu relevanten Nebenwirkungen. Daher besteht großes Interesse, neue Behandlungsstrategien zu entwickeln. Basierend auf anekdotischen Berichten und kleinen Studien, in denen über positive Effekte durch Cannabis und cannabisbasierte Medikamente bei Patienten mit GTS berichtet wurde, wird in kontrollierten Studien untersucht, ob Medikamente, die durch unterschiedliche Mechanismen zu einer Stimulation des Endocannabinoidsystems führen, wirksam und verträglich in der Therapie von Tics und Komorbiditäten sind. In dieser Übersicht werden bisherige Studienergebnisse vorgestellt. Es werden Hypothesen diskutiert, warum Cannabinoide in der Behandlung des GTS wirksam sein könnten. Schließlich werden praktische Empfehlungen zur Therapie mit cannabisbasierten Medikamenten gegeben.

Introduction: Speech dysfluency, often referred to as stuttering, is a frequent speech disorder encountered in about 5% of children. Although in the majority of people affected, symptoms improve in adulthood, in some patients, stuttering persists and significantly impairs everyday functioning and quality of life. Treatment for stuttering includes speech therapy, cognitive behavioral therapy, and relaxation techniques. However, a substantial number of patients do not benefit sufficiently from these treatment strategies or are even treatment resistant. Methods: We present the case of a 20-year-old male with treatment-resistant stuttering, who markedly improved after treatment with medicinal cannabis. Results: Besides improved speech fluency as assessed by several phoniatric tests, we observed remission of (social) anxiety, improved mood, and reduced stress, resulting in an overall improvement of quality of life after cannabis therapy. The patient, in addition, reported improved attention, concentration, and sleep, increased self-confidence, and better social life. No side effects occurred. Over a time period of more than a year, treatment was equally effective. In an interview, the patient describes his personal view and the influence of cannabis-based treatment on his life. Conclusions: Medicinal cannabis could be effective in treatment of refractory stuttering, but these preliminary data have to be confirmed in controlled studies.

Introduction Gilles de la Tourette syndrome (GTS) is a childhood onset disorder characterised by the presence of motor and vocal tics. The guidelines of both the American Academy of Neurology (AAN) as well as the European Society for the Study of Tourette Syndrome (ESSTS) recommend behavioural therapy and pharmacotherapy, mainly with antipsychotics, as first line treatments for tics. In spite of these well-established therapeutic approaches, a significant number of patients are dissatisfied because of insufficient tic reduction or intolerable side effects. Previous studies have suggested that cannabis-based medicine (CBM) might be an alternative treatment in these patients. Material and methods Two reviewers (KS, NS) searched the electronic database of PubMed on 1 July, 2021 for relevant studies using the search terms: ('Tourette syndrome' [MeSH Terms] OR 'Gilles de la Tourette syndrome' [MeSH Terms] OR 'tic disorders' [MeSH Terms] OR 'tics' [MeSH Terms] OR 'tic disorders'[Title/Abstract]) AND ('cannabis-based medicine' [Title/Abstract] OR 'cannabis' [Title/Abstract] OR 'dronabinol' [Title/Abstract] OR 'nabiximols' [Title/Abstract] OR 'tetrahydrocannabinol' [Title/Abstract] OR 'THC' [Title/Abstract] OR 'cannabidiol' [Title/Abstract], limit: 'humans'. These studies were further reviewed for additional relevant citations. The titles and abstracts of the studies obtained through this search were examined by two reviewers (KS, NS) in order to determine article inclusion. Discrepancies were addressed by the reviewers through discussion and eventually conversation with the senior reviewer (KMV). Results Although the amount of evidence supporting the use of CBM in GTS is growing, the majority of studies are still limited to case reports, case series, and open uncontrolled studies. To date, only two small randomised controlled trials (RCTs) using tetrahydrocannabinol (THC, dronabinol) have been published demonstrating the safety and efficacy of this intervention in the treatment of tics in patients with GTS. On the other hand, another RCT with Lu AG06466 (formerly known as ABX-1431), a modulator of endocannabinoid neurotransmission, has failed to prove effective in the therapy of GTS. Accordingly, under the guidelines of both the ESSTS and the AAN, treatment with CBM is categorised as an experimental intervention that should be applied to patients who are otherwise treatment-resistant. Conclusions Increasing evidence suggests that CBM is efficacious in the treatment of tics and psychiatric comorbidities in patients with GTS. The results of ongoing larger RCTs, such as CANNA-TICS (ClinicalTrials.gov Identifier: NCT03087201), will further clarify the role of CBM in the treatment of patients with GTS.

Introduction: Gilles de la Tourette syndrome (GTS) is a neuropsychiatric disorder that is characterized by motor and vocal tics and psychiatric comorbidities, including attention deficit/hyperactivity disorder (ADHD) and obsessive-compulsive behavior/disorder (OCB/OCD). From anecdotal reports and preliminary controlled studies, it is suggested that cannabis-based medicine (CBM) may improve tics and comorbidities in adults with GTS. This study was designed to further investigate efficacy and safety of CBM in GTS and specifically compare effects of different CBM. Materials and Methods: First, we performed a retrospective data analysis including all those adult patients seen at our clinic, who had used CBM for the treatment of GTS at some time. All these patients were asked to complete an online survey (second study part) to receive more detailed data about treatment with CBM. Results: From medical records, we identified 98 patients who had used CBM (most often street cannabis followed by nabiximols, dronabinol, medicinal cannabis) for the treatment of GTS: Of the 38 patients who were able to judge, 66% preferred treatment with medicinal cannabis, 18% dronabinol, 11% nabiximols, and 5% street cannabis. Altogether, CBM resulted in a subjective improvement of tics (of about 60% in 85% of treated cases), comorbidities (55% of treated cases, most often OCB/OCD, ADHD, and sleeping disorders), and quality of life (93%). The effects of CBM appear to persist in the long term. Adverse events occurred in half of the patients, but they were rated as tolerable. Dosages of all CBM varied markedly. Patients assessed cannabis (with a preference for tetrahydrocannabinol [THC]-rich strains) as more effective and better tolerated compared with nabiximols and dronabinol. These data were confirmed by results obtained from the online survey (n=40). Conclusion: From our results, it is further supported that CBM might be effective and safe in the treatment of tics and comorbidities at least in a subgroup of adult patients with GTS. In our sample, patients favored THC-rich cannabis over dronabinol and nabiximols, which might be related to the entourage effect of cannabis. However, several limitations of the study have to be taken into considerations such as the open uncontrolled design and the retrospective data analysis.

Fusobacterium nucleatum is a common oral bacterium and a major producer of H2S, a toxic gas linked to the pathogenesis of periodontal disease. The bacterium encodes a fold type II pyridoxal l-phosphate (PLP)-dependent enzyme, Fn1220 or lanthionine synthase (LS), that generates H2S and l-lanthionine (a component of the peptidoglycan layer) through β-replacement of l-cysteine by a second molecule of l-cysteine. Herein, we show through detailed kinetic analysis that LS elicits catalytic promiscuity as demonstrated for other fold type II PLP-dependent homologues, namely, O-acetylserine sulfhydrylase (OASS) and cystathionine β-synthase (CBS). Like OASS, LS can assimilate H2S by catalyzing the β-replacement of O-acetyl-l-serine by sulfide to form l-cysteine. However, the turnover for this reaction in LS is slower than that of other studied OASS enzymes due to slower conversion to the α-aminoacrylate intermediate. Similar to yeast and human CBS, LS can generate H2S and l-cystathionine through β-replacement of l-cysteine by a second molecule of l-homocysteine; however, whereas this is the main H2S-forming reaction in CBS, it is not for LS. LS shows a marked preference for forming H2S and l-lanthionine through the condensation of 2 equiv of l-cysteine. Sequence alignment of LS with other CBS and OASS enzymes and inspection of the LS crystal structure in the external aldimine state with l-lanthionine reveal that LS possesses a unique loop that engages in hydrogen-bond contact with the product, providing a structural rationale for the enzyme's catalytic preference for H2S and l-lanthionine biosynthesis.

Flavodoxins are small flavin mononucleotide (FMN)‐containing proteins that mediate a variety of electron transfer processes. The primary sequence of flavodoxin from Fusobacterium nucleatum, a pathogenic oral bacterium, is marked with a number of distinct features including a glycine to lysine (K13) substitution in the highly conserved phosphate‐binding loop (T/S‐X‐T‐G‐X‐T), variation in the aromatic residues that sandwich the FMN cofactor, and a more even distribution of acidic and basic residues. The E (ox/sq) (oxidized/semiquinone; −43 mV) and E (sq/hq) (semiquinone/hydroquinone; −256 mV) are the highest recorded reduction potentials of known long‐chain flavodoxins. These more electropositive values are a consequence of the apoprotein binding to the FMN hydroquinone anion with ~70‐fold greater affinity compared to the oxidized form of the cofactor. Inspection of the FnFld crystal structure revealed the absence of a hydrogen bond between the protein and the oxidized FMN N5 atom, which likely accounts for the more electropositive E (ox/sq). The more electropositive E (sq/hq) is likely attributed to only one negatively charged group positioned within 12 Å of the FMN N1. We show that natural substitutions of highly conserved residues partially account for these more electropositive reduction potentials.

Preeclampsia is a hypertensive disorder of pregnancy characterized by maternal endothelial dysfunction and end-organ damage. The antiangiogenic factor, sFlt-1 (soluble FMS-like tyrosine kinase-1) has been strongly implicated in the pathogenesis of preeclampsia. sFlt-1 is secreted into the maternal circulation where it antagonizes VEGF (vascular endothelial growth factor) and ultimately disrupts vascular homeostasis. However, the upstream mechanisms regulating release of sFlt-1 are poorly characterized. We investigated the roles of key prosurvival pathways, EGFR (epidermal growth factor receptor) signaling, and the mitochondria, in regulating sFlt-1 production. We initially found that the mRNA and protein of EGFR and downstream adaptor molecules were significantly increased in preeclamptic placental tissue relative to normotensive controls. Inhibiting the EGFR signaling cascade using siRNA (small interfering ribonucleic acid) or small molecule inhibitors significantly reduced sFlt-1 release from primary cytotrophoblast (placental cells). Additionally, inhibiting the mitochondrial electron transport chain or activating downstream energy-sensing molecules (AMPK [AMP-activated kinase], SIRT1 [sirtuin 1 ], and PGC1α [PPAR-γ co-activator 1]) also significantly reduced sFlt-1 secretion from cytotrophoblast cells, without affecting EGFR signaling. In vivo, treating pregnant mice with gefitinib (an EGFR inhibitor) or resveratrol (perturbs mitochondrial function) significantly reduced circulating murine sFlt-1 compared with vehicle control. Furthermore, treating primary cytotrophoblasts with therapeutics which have been previously found to reduce sFlt-1 secretion, either reduced EGFR signaling (esomeprazole and statins) or perturbed mitochondrial function (esomeprazole, resveratrol, and metformin). Additionally, targeting both pathways simultaneously produced additive reductions in sFlt-1 secretion. Thus, we have identified 2 key survival pathways that seem to be overactive in preeclampsia and involved in the regulation of placental sFlt-1 secretion.

Episodic memory requires information to be stored and recalled in sequential order, and these processes are disrupted in schizophrenia. Hippocampal phase precession and theta sequences are thought to provide a biological mechanism for sequential ordering of experience at timescales suitable for plasticity. These phenomena have not previously been examined in any models of schizophrenia risk. Here, we examine these phenomena in a maternal immune activation (MIA) rodent model. We show that while individual pyramidal cells in the CA1 region continue to precess normally in MIA animals, the starting phase of precession as an animal enters a new place field is considerably more variable in MIA animals than in controls. A critical consequence of this change is a disorganization of the ordered representation of experience via theta sequences. These results provide the first evidence of a biological-level mechanism that, if it occurs in schizophrenia, may explain aspects of disorganized sequential processing that contribute to the cognitive symptoms of the disorder. SIGNIFICANCE STATEMENT Hippocampal phase precession and theta sequences have been proposed as biophysical mechanisms by which the sequential structure of cognition might be ordered. Disturbances of sequential processing have frequently been observed in schizophrenia. Here, we show for the first time that phase precession and theta sequences are disrupted in a maternal immune activation (MIA) model of schizophrenia risk. This is a result of greater variability in the starting phase of precession, indicating that the mechanisms that coordinate precession at the assembly level are disrupted. We propose that this disturbance in phase precession underlies some of the disorganized cognitive symptoms that occur in schizophrenia. These findings could have important preclinical significance for the identification and treatment of schizophrenia risk factors.

The few established causal genes in Alzheimer’s disease (AD), mutations in APP and PSENs, have been functionally characterized using biomarkers, capturing an in vivo profile reflecting the disease’s initial preclinical phase. SORL1, a gene encoding the endosome recycling receptor SORLA, epidemiologically behaves as a causal gene when truncating mutations lead to partial loss of protein function. Here, in an effort to test whether SORL1 can indeed function as an AD causal gene, we used CRISPR-Cas9-based gene editing to develop a novel model of SORL1 haploinsufficiency in Gottingen Minipigs taking advantage of porcine models for biomarker investigations. SORL1 haploinsufficiency in young minipigs was found to phenocopy the preclinical in vivo profile of AD observed with other causal genes, resulting in spinal fluid abnormalities in Ab and tau, with no evident neurodegeneration or amyloid plaque formation. These studies provide functional support that SORL1 is a bona fide causal gene in AD, and when taken together with recent insight on other AD-causal genes, support the idea that dysfunctional endosomal recycling is a dominant pathogenic pathway in the disease.

Tsetsos, Fotis et al., Tourette syndrome (TS) is a neuropsychiatric disorder of complex genetic architecture involving multiple interacting genes. Here, we sought to elucidate the pathways that underlie the neurobiology of the disorder through genome-wide analysis. We analyzed genome-wide genotypic data of 3581 individuals with TS and 7682 ancestry-matched controls and investigated associations of TS with sets of genes that are expressed in particular cell types and operate in specific neuronal and glial functions. We employed a self-contained, set-based association method (SBA) as well as a competitive gene set method (MAGMA) using individual-level genotype data to perform a comprehensive investigation of the biological background of TS. Our SBA analysis identified three significant gene sets after Bonferroni correction, implicating ligand-gated ion channel signaling, lymphocytic, and cell adhesion and transsynaptic signaling processes. MAGMA analysis further supported the involvement of the cell adhesion and trans-synaptic signaling gene set. The lymphocytic gene set was driven by variants in FLT3, raising an intriguing hypothesis for the involvement of a neuroinflammatory element in TS pathogenesis. The indications of involvement of ligand-gated ion channel signaling reinforce the role of GABA in TS, while the association of cell adhesion and trans-synaptic signaling gene set provides additional support for the role of adhesion molecules in neuropsychiatric disorders. This study reinforces previous findings but also provides new insights into the neurobiology of TS., This research is co-financed by Greece and the European Union (European Social Fund—ESF) through the Operational Programme «Human Resources Development, Education and Lifelong Learning» in the context of the project “Reinforcement of Postdoctoral Researchers - 2nd Cycle” (MIS-5033021), implemented by the State Scholarships Foundation (IKY). L.K.D. was supported by grants from the National Institutes of Health including U54MD010722-04, R01NS102371, R01MH113362, U01HG009086, R01MH118223, DP2HD98859, R01DC16977, R01NS105746, R56MH120736, R21HG010652, and RM1HG009034.

Background: FSHD is caused by specific genetic mutations resulting in activation of the Double Homeobox 4 gene (DUX4). DUX4 targets hundreds of downstream genes eventually leading to muscle atrophy, oxidative stress, abnormal myogenesis, and muscle inflammation. We hypothesized that DUX4-induced aberrant expression of genes triggers a sustained autoimmune response against skeletal muscle cells. Objective: This study aimed at the identification of autoantibodies directed against muscle antigens in FSHD. Moreover, a possible relationship between serum antibody reactivity and DUX4 expression was also investigated. Methods: FSHD sera (N = 138, 48±16 years, 48% male) and healthy control sera (N = 20, 47±14 years, 50% male) were analyzed by immunoblotting for antibodies against several skeletal muscle protein extracts: healthy muscle, FSHD muscle, healthy and FSHD myotubes, and inducible DUX4 expressing myoblasts. In addition, DUX4 expressing myoblasts were analyzed by immunofluorescence with FSHD and healthy control sera. Results: The results showed that the reactivity of FSHD sera did not significantly differ from that of healthy controls, with all the tested muscle antigen extracts. Besides, the immunofluorescent staining of DUX4-expressing myoblasts was not different when incubated with either FSHD or healthy control sera. Conclusion: Since the methodology used did not lead to the identification of disease-specific autoantibodies in the FSHD cohort, we suggest that autoantibody-mediated pathology may not be an important disease mechanism in FSHD. Nevertheless, it is crucial to further unravel if and which role the immune system plays in FSHD pathogenesis. Other innate as well as adaptive immune players could be involved in the complex DUX4 cascade of events and could become appealing druggable targets.

Antibiotic resistance has become a global threat. In addition to acquiring resistance via horizontal gene transfer, bacteria can evade killing by temporarily modifying their cell envelope to prevent antibiotic-bacterial interactions. A critical gap in knowledge is how bacteria balance the metabolic needs of altering the cell envelope with the constant need to generate energy. Cross-regulation between two signaling networks in Escherichia coli increases resistance to positively charged antibiotics. We show that increased resistance is supported by metabolic re-wiring controlled by the QseB transcription factor. QseB controls the increase in 2-oxoglutarate required for lipid A modification, by upregulating three anaplerotic pathways that feed acetyl Co-A, succinate and fumarate into the TCA cycle. Exogenous addition of 2-oxoglutarate restores antibiotic resistance in the qseB deletion mutant. Antibiotic resistant clinical isolates bear mutations within QseB-mediated anaplerotic pathways. These findings are significant, because they uncover a previously unknown mechanism of metabolic control of antibiotic resistance.

Since popular microbial cell factories have typically not been selected for efficient respiratory energy coupling, their ATP yields from sugar catabolism are often suboptimal. In aerobic industrial processes, suboptimal energy coupling results in reduced product yields on sugar, increased process costs for oxygen transfer, and volumetric productivity limitations due to limitations in gas transfer and cooling. This study provides insights into the contribution of mechanisms of respiratory energy coupling in the yeast cell factory Ogataea parapolymorpha under different growth conditions and provides a basis for rational improvement of energy coupling in yeast cell factories. Analysis of energy metabolism of O. parapolymorpha at extremely low specific growth rates indicated that this yeast reduces its energy requirements for cellular maintenance under extreme energy limitation. Exploration of the mechanisms for this increased energetic efficiency may contribute to an optimization of the performance of industrial processes with slow-growing eukaryotic cell factories., The thermotolerant yeast Ogataea parapolymorpha (formerly Hansenula polymorpha) is an industrially relevant production host that exhibits a fully respiratory sugar metabolism in aerobic batch cultures. NADH-derived electrons can enter its mitochondrial respiratory chain either via a proton-translocating complex I NADH-dehydrogenase or via three putative alternative NADH dehydrogenases. This respiratory entry point affects the amount of ATP produced per NADH/O2 consumed and therefore impacts the maximum yield of biomass and/or cellular products from a given amount of substrate. To investigate the physiological importance of complex I, a wild-type O. parapolymorpha strain and a congenic complex I-deficient mutant were grown on glucose in aerobic batch, chemostat, and retentostat cultures in bioreactors. In batch cultures, the two strains exhibited a fully respiratory metabolism and showed the same growth rates and biomass yields, indicating that, under these conditions, the contribution of NADH oxidation via complex I was negligible. Both strains also exhibited a respiratory metabolism in glucose-limited chemostat cultures, but the complex I-deficient mutant showed considerably reduced biomass yields on substrate and oxygen, consistent with a lower efficiency of respiratory energy coupling. In glucose-limited retentostat cultures at specific growth rates down to ∼0.001 h−1, both O. parapolymorpha strains showed high viability. Maintenance energy requirements at these extremely low growth rates were approximately 3-fold lower than estimated from faster-growing chemostat cultures, indicating a stringent-response-like behavior. Quantitative transcriptome and proteome analyses indicated condition-dependent expression patterns of complex I subunits and of alternative NADH dehydrogenases that were consistent with physiological observations. IMPORTANCE Since popular microbial cell factories have typically not been selected for efficient respiratory energy coupling, their ATP yields from sugar catabolism are often suboptimal. In aerobic industrial processes, suboptimal energy coupling results in reduced product yields on sugar, increased process costs for oxygen transfer, and volumetric productivity limitations due to limitations in gas transfer and cooling. This study provides insights into the contribution of mechanisms of respiratory energy coupling in the yeast cell factory Ogataea parapolymorpha under different growth conditions and provides a basis for rational improvement of energy coupling in yeast cell factories. Analysis of energy metabolism of O. parapolymorpha at extremely low specific growth rates indicated that this yeast reduces its energy requirements for cellular maintenance under extreme energy limitation. Exploration of the mechanisms for this increased energetic efficiency may contribute to an optimization of the performance of industrial processes with slow-growing eukaryotic cell factories.

Although several lines of evidence support the hypothesis of a dysregulation of serotoninergic neurotransmission in the pathophysiology of obsessive-compulsive disorder (OCD), there is also evidence for an involvement of other pathways such as the GABAergic, glutamatergic, and dopaminergic systems. Only recently, data obtained from a small number of animal studies alternatively suggested an involvement of the endocannabinoid system in the pathophysiology of OCD reporting beneficial effects in OCD-like behavior after use of substances that stimulate the endocannabinoid system. In humans, until today, only two case reports are available reporting successful treatment with dronabinol (tetrahydrocannabinol, THC), an agonist at central cannabinoid CB1 receptors, in patients with otherwise treatment refractory OCD. In addition, data obtained from a small open uncontrolled trial using the THC analogue nabilone suggest that the combination of nabilone plus exposure-based psychotherapy is more effective than each treatment alone. These reports are in line with data from a limited number of case studies and small controlled trials in patients with Tourette syndrome (TS), a chronic motor and vocal tic disorder often associated with comorbid obsessive compulsive behavior (OCB), reporting not only an improvement of tics, but also of comorbid OCB after use of different kinds of cannabis-based medicines including THC, cannabis extracts, and flowers. Here we present the case of a 22-year-old male patient, who suffered from severe OCD since childhood and significantly improved after treatment with medicinal cannabis with markedly reduced OCD and depression resulting in a considerable improvement of quality of life. In addition, we give a review of current literature on the effects of cannabinoids in animal models and patients with OCD and suggest a cannabinoid hypothesis of OCD.

While the importance of the serotonergic system in obsessive compulsive disorder (OCD) is well established, its role in Tourette syndrome (TS) is uncertain. Particularly in TS patients with comorbid OCD (TS + OCD), decreased serotonin transporter (SERT) binding has been suggested. Here, we investigated for the first time SERT binding in TS patients with and without OCD (TS - OCD) compared to both healthy controls (HC) and OCD patients as well as the influence of escitalopram using the potent SERT imaging ligand [

Amyris is a fermentation product company that leverages synthetic biology and has been bringing novel fermentation products to the market since 2009. Driven by breakthroughs in genome editing, strain construction and testing, analytics, automation, data science, and process development, Amyris has commercialized nine separate fermentation products over the last decade. This has been accomplished by partnering with the teams at 17 different manufacturing sites around the world. This paper begins with the technology that drives Amyris, describes some key lessons learned from early scale-up experiences, and summarizes the technology transfer procedures and systems that have been built to enable moving more products to market faster. Finally, the breadth of the Amyris product portfolio continues to expand; thus the steps being taken to overcome current challenges (e.g. automated strain engineering can now outpace the rest of the product commercialization timeline) are described.

The neurotrophin GDNF guides development of the enteric nervous system (ENS) in embryogenesis and directs survival and axon outgrowth in postnatal myenteric neurons in vitro. GDNF expression in intestinal smooth muscle cells is dynamic, with upregulation by inflammatory cytokines in vitro or intestinal inflammation in vivo, but the role of post-translational proteolytic cleavage is undefined. In a co-culture model of myenteric neurons, smooth muscle and glia, inhibition of serine or cysteine protease activity was ineffective against the >2-fold increase in axon density caused by TNFα. However, inhibitors of metalloproteinases (MMP) identified an essential role of MMP-9, and qPCR and western blotting showed that pro-inflammatory cytokines increased both mRNA and protein expression for MMP-9, in both cellular lysates and conditioned medium (CM). Inhibition of MMP-9 prevented the cytokine-induced increase in mature GDNF in CM or cellular lysates of co-cultures or cell lines of intestinal smooth muscle cells (ISMC) from adult rat colon. Western blotting showed parallel upregulation of mature GDNF and MMP-9 vs control in ISMC isolated on Day 2 of TNBS-induced colitis. Nonetheless, transfection of GDNF plasmid into HEK-293 cells as a carrier system, or directly into the co-culture model, conveyed a strong neurotrophic effect that was MMP-9 dependent. We conclude that MMP-9 activity is required for the neurotrophic effects of GDNF on myenteric neurons in vitro. However, the coordinated upregulation of GDNF and MMP-9 in intestinal smooth muscle by inflammatory cytokines provides a supportive, target cell-derived environment that limits inflammatory damage to the ENS.

Gilles de la Tourette syndrome (GTS) is a neuropsychiatric disorder characterized by motor and vocal tics. There are several hypotheses as to the cause of the disease. One of which suggests that the immune system is involved in the pathophysiology of GTS. Here, we present a 40-year-old female patient with a typical history and clinical presentation of GTS with tics and psychiatric comorbidities, in whom positive oligoclonal bands (OCBs) type 2 in cerebral spinal fluid (CSF) were detected in an earlier study. At that time point, all other investigations were unremarkable (including neurological examination and cMRI), but 2 years later, she presented with further neurological symptoms including tetraparesis mostly affecting the left limbs, distal hypesthesia and paresthesia, and dyspnea. Since further examinations (including EMG, MRI, CSF, virological, and bacteriological tests, as well as autoimmune-encephalitis antibodies) revealed normal results, based on clinical presentation, the diagnosis of Guillain-Barre-like immune-mediated neuropathy was made and treatment with intravenous immunoglobulins (IVIg) (30 g/day for 5 days) was initiated resulting in complete remission of immune-mediated neuropathy. Based on the "immune hypothesis" of GTS, we were interested in whether in this patient positive CSF OCBs might serve as a biomarker for treatment response of tics and additional GTS-related psychiatric symptoms to IVIg, and therefore assessed tics, premonitory urges, and psychiatric comorbidities before and several times after the IVIg treatment. However, even though immune-mediated neuropathy resolved after treatment, tics, premonitory urges, and comorbidities remained unchanged. Thus, this case study suggests that treatment with IVIg is not effective in the treatment of GTS-even in a patient with intrathecal antibody synthesis as expressed by CSF isolated OCBs.

This study used high throughput, image-based phenotyping (HTP) to distinguish growth patterns, detect facilitation and interpret variations to nutrient uptake in a model mixed-pasture system in response to factorial low and high nitrogen (N) and phosphorus (P) application. HTP has not previously been used to examine pasture species in mixture. We used red-green-blue (RGB) imaging to obtain smoothed projected shoot area (sPSA) to predict absolute growth (AG) up to 70 days after planting (sPSA, DAP 70), to identify variation in relative growth rates (RGR, DAP 35–70) and detect overyielding (an increase in yield in mixture compared with monoculture, indicating facilitation) in a grass-legume model pasture. Finally, using principal components analysis we interpreted between species changes to HTP-derived temporal growth dynamics and nutrient uptake in mixtures and monocultures. Overyielding was detected in all treatments and was driven by both grass and legume. Our data supported expectations of more rapid grass growth and augmented nutrient uptake in the presence of a legume. Legumes grew more slowly in mixture and where growth became more reliant on soil P. Relative growth rate in grass was strongly associated with shoot N concentration, whereas legume RGR was not strongly associated with shoot nutrients. High throughput, image-based phenotyping was a useful tool to quantify growth trait variation between contrasting species and to this end is highly useful in understanding nutrient-yield relationships in mixed pasture cultivations.

Genetic studies in Tourette syndrome (TS) are characterized by scattered and poorly replicated findings. We aimed to replicate findings from candidate gene and genome-wide association studies (GWAS). Our cohort included 465 probands with chronic tic disorder (93% TS) and both parents from 412 families (some probands were siblings). We assessed 75 single nucleotide polymorphisms (SNPs) in 465 parent–child trios; 117 additional SNPs in 211 trios; and 4 additional SNPs in 254 trios. We performed SNP and gene-based transmission disequilibrium tests and compared nominally significant SNP results with those from a large independent case–control cohort. After quality control 71 SNPs were available in 371 trios; 112 SNPs in 179 trios; and 3 SNPs in 192 trios. 17 were candidate SNPs implicated in TS and 2 were implicated in obsessive–compulsive disorder (OCD) or autism spectrum disorder (ASD); 142 were tagging SNPs from eight monoamine neurotransmitter-related genes (including dopamine and serotonin); 10 were top SNPs from TS GWAS; and 13 top SNPs from attention-deficit/hyperactivity disorder, OCD, or ASD GWAS. None of the SNPs or genes reached significance after adjustment for multiple testing. We observed nominal significance for the candidate SNPs rs3744161 (TBCD) and rs4565946 (TPH2) and for five tagging SNPs; none of these showed significance in the independent cohort. Also, SLC1A1 in our gene-based analysis and two TS GWAS SNPs showed nominal significance, rs11603305 (intergenic) and rs621942 (PICALM). We found no convincing support for previously implicated genetic polymorphisms. Targeted re-sequencing should fully appreciate the relevance of candidate genes. Electronic supplementary material The online version of this article (doi:10.1007/s00406-017-0808-8) contains supplementary material, which is available to authorized users.

Early acting cytokines and growth factors such as those of the CD131 βc subunit, may offer an alternative method to the current use of antibiotics and chemicals such as anthelmintics in maintaining Porcine (Po) health. Thus far, the recombinant Po (rPo) Granulocyte-macrophage colony-stimulating factor (GM-CSF), rPo interleukin-3 (IL-3) and rPo interleukin-5 (IL-5) proteins have been identified and cloned and the biological activity of each cytokine has been confirmed in vitro, however, in vivo immune system regulation and hematopoietic stem cell (HSC) augmentation are regulated by numerous cytokines and cellular signals within the bone marrow (BM) niche. In order to quantify the use of recombinant cytokines in augmenting the immune response, it is necessary to determine the stages of hematopoiesis induced by each cytokine and possible areas of synergy requiring further investigation. Here we used the chemotherapeutic agent 5-fluorouracil (5-FU), to chemically induce a state of myelosuppression in young pigs. This allowed for the monitoring of both the autologous BM reconstitution and recombinant cytokine induced BM repopulation, precursor cell proliferation and cellular differentiation. The recombinant cytokines PoGM-CSF, PoIL-3 and PoIL-5 were administered by intramuscular injections (i.m.) following confirmation of 5-FU induced leukocytopenia. Blood and BM samples were collected and then analysed for cell composition. Statistically significant results were observed in several blood cell populations including eosinophils for animals treated with rPoIL-5, rPoGM-CSF and basophils for animals treated with rPoIL-3. BM analysis of CD90+ and CD172a+ cells confirmed myelosuppression in week one with significant results observed between rPoIL-3 and the 5-FU control group in week two and for the rPoGM-CSF group in week three. These results have demonstrated the effects of each of these rPo cytokines within the hematopoietic processes of the pig and may demonstrate similar outcomes in other mammalian models including human.

Vibrio fischeri is a bacterial symbiont that colonizes the light organ of the Hawaiian bobtail squid, Euprymna scolopes. Certain strains of V. fischeri express a type VI secretion system (T6SS), which delivers effectors into neighboring cells that result in their death. Strains that are susceptible to the T6SS fail to establish symbiosis with a T6SS-positive strain within the same location of the squid light organ, which is a phenomenon termed strain incompatibility. This study investigates the regulation of the T6SS in V. fischeri strain FQ-A001. Here, we report that the expression of Hcp, a necessary structural component of the T6SS, depends on the alternative sigma factor σ(54) and the bacterial enhancer binding protein VasH. VasH is necessary for FQ-A001 to kill other strains, suggesting that VasH-dependent regulation is essential for the T6SS of V. fischeri to affect intercellular interactions. In addition, this study demonstrates VasH-dependent transcription of hcp within host-associated populations of FQ-A001, suggesting that the T6SS is expressed within the host environment. Together, these findings establish a model for transcriptional control of hcp in V. fischeri within the squid light organ, thereby increasing understanding of how the T6SS is regulated during symbiosis. IMPORTANCE Animals harbor bacterial symbionts with specific traits that promote host fitness. Mechanisms that facilitate intercellular interactions among bacterial symbionts impact which bacterial lineages ultimately establish symbiosis with the host. How these mechanisms are regulated is poorly characterized in nonhuman bacterial symbionts. This study establishes a model for the transcriptional regulation of a contact-dependent killing machine, thereby increasing understanding of mechanisms by which different strains compete while establishing symbiosis.

Untreated microangiopathic hemolytic anemia in pregnancy is associated with adverse maternal and perinatal outcomes. Accurate diagnosis is challenging owing to nonspecific clinical features and pathologic findings. Timely initiation of appropriate management is essential to optimize maternal and perinatal outcomes.A 26-year-old primiparous woman presented at 20 weeks of gestation with new-onset microangiopathic hemolytic anemia on a background of poorly controlled type 1 diabetes. She received eculizumab for presumed atypical hemolytic uremic syndrome. At 24 weeks of gestation, she developed superimposed early-onset preeclampsia; she delivered at 27 weeks of gestation after continuing eculizumab.Eculizumab may prolong pregnancy in early-onset preeclampsia. Additional research is needed to assess short-term and long-term maternal and newborn outcomes.

Background: Several lines of evidence support the hypothesis of an autoimmune origin of Gilles de la Tourette-Syndrome (GTS). Accordingly, in a recent study we detected positive oligoclonal bands (OCB) in cerebrospinal fluid (CSF) in >30% of adult patients indicating an intrathecal antibody synthesis. However, until today no corresponding antibodies could be identified. The aims of this study were to replicate our findings of positive OCB in an independent sample and to detect CSF autoantibodies. Methods: In this prospective study, 20 adult patients with GTS (male: female = 18:2, median age 36.1 years ± 14.34 SD) were included. All patients were thoroughly clinically characterized. Magnetic Resonance Imaging (MRI) and CSF standard measurements were performed. Isoelectric focusing on polyacrylamide gels with silver staining was used to detect OCB. To examine specific and unspecified autoantibodies, we used transfected Human Embryonic Kidney (HEK) cells expressing different surface antigens (NMDA-, CASPR2-, LGI1-, AMPA-, or GABAB1/B), indirect immunofluorescence on different brain tissue sections, and enzyme-linked visualization. Additionally, we differentiated Glioma stem cells SY5Y (human neuroblastoma) using retinoic acid and astrocytes (rat). Results: CSF analyses showed positive OCB (type 2) in 4/20 patients (20%). Using transfected HEK cells we did not find specific surface-autoantibodies. Immunohistochemistry on tissue-sections, SY5Y Glioma stem-cells, and astrocytes showed no specific binding patterns either. Conclusions: Our results corroborate previous findings and demonstrate positive OCB in a substantial number of patients with GTS (prevalence in healthy controls: 5%). Although this is the largest study investigating CSF autoantibodies in GTS using several techniques, we failed to detect any specific or unspecified autoantibodies.

Bacteria that have the capacity to fill the same niche will compete with one another for the space and resources available within an ecosystem. Such competition is heightened among different strains of the same bacterial species. Nevertheless, different strains often inhabit the same host. The molecular mechanisms that impact competition between different strains within the same host are poorly understood. To address this knowledge gap, the type VI secretion system (T6SS), which is a mechanism for bacteria to kill neighboring cells, was examined in the marine bacterium Vibrio fischeri. Different strains of V. fischeri naturally colonize the light organ of the bobtail squid Euprymna scolopes. The genome of FQ-A001, a T6SS-positive strain, features two hcp genes that are predicted to encode identical subunits of the T6SS. Coincubation assays showed that either hcp gene is sufficient for FQ-A001 to kill another strain via the T6SS in vitro. Additionally, induction of hcp expression is sufficient to induce killing activity in an FQ-A001 mutant lacking both hcp genes. Squid colonization assays involving inocula of FQ-A001-derived strains mixed with ES114 revealed that both hcp genes must be deleted for FQ-A001 and ES114 to occupy the same space within the light organ. These experimental results provide insight into the genetic factors necessary for the T6SS of V. fischeri to function in vivo, thereby increasing understanding of the molecular mechanisms that impact strain diversity within a host. IMPORTANCE Different bacterial strains compete to occupy the same niche. The outcome of such competition can be affected by the type VI secretion system (T6SS), an intercellular killing mechanism of bacteria. Here an animal-bacterial symbiosis is used as a platform for study of the genetic factors that promote the T6SS-mediated killing of one strain by another. Identification of the molecular determinants of T6SS function in vivo contributes to the understanding of how different strains interact within a host.

SummarySex differences in growth rate in very early embryos have been recognized in a variety of mammals and attributed to sex-chromosome complement effects as they occur before overt sexual differentiation. We previously found that sex-chromosome complement, rather than sex hormones regulates heterochromatin-mediated silencing of a transgene and autosomal gene expression in mice. Here, sex dimorphism in proliferation was investigated. We confirm that male embryonic fibroblasts proliferate faster than female fibroblasts and show that this proliferation advantage is completely dependent upon heterochromatin protein 1 gamma (HP1γ). To determine whether this sex-regulatory effect of HP1γ was a more general phenomenon, we performed RNA sequencing on MEFs derived from males and females, with or without HP1γ. Strikingly, HP1γ was found to be crucial for regulating nearly all sexually dimorphic autosomal gene expression because deletion of the HP1γ gene in males abolished sex differences in autosomal gene expression. The identification of a key epigenetic modifier as central in defining gene expression differences between males and females has important implications for understanding physiological sex differences and sex bias in disease.

Objective: Tourette's syndrome is polygenic and highly heritable. Genome-wide association study (GWAS) approaches are useful for interrogating the genetic architecture and determinants of Tourette's syndrome and other tic disorders. The authors conducted a GWAS meta-analysis and probed aggregated Tourette's syndrome polygenic risk to test whether Tourette's and related tic disorders have an underlying shared genetic etiology and whether Tourette's polygenic risk scores correlate with worst-ever tic severity and may represent a potential predictor of disease severity.Methods: GWAS meta-analysis, gene-based association, and genetic enrichment analyses were conducted in 4,819 Tourette's syndrome case subjects and 9,488 control subjects. Replication of top loci was conducted in an independent population-based sample (706 case subjects, 6,068 control subjects). Relationships between Tourette's polygenic risk scores (PRSs), other tic disorders, ascertainment, and tic severity were examined.Results: GWAS and gene-based analyses identified one genome-wide significant locus within FLT3 on chromosome 13, rs2504235, although this association was not replicated in the population-based sample. Genetic variants spanning evolutionarily conserved regions significantly explained 92.4% of Tourette's syndrome heritability. Tourette'sassociated genes were significantly preferentially expressed in dorsolateral prefrontal cortex. Tourette's PRS significantly predicted both Tourette's syndrome and tic spectrum disorders status in the population-based sample. Tourette's PRS also significantly correlated with worst-ever tic severity and was higher in case subjects with a family history of tics than in simplex case subjects.Conclusions: Modulation of gene expression through noncoding variants, particularly within cortico-striatal circuits, is implicated as a fundamental mechanism in Tourette's syndrome pathogenesis. At a genetic level, tic disorders represent a continuous spectrum of disease, supporting the unification of Tourette's syndrome and other tic disorders in future diagnostic schemata. Tourette's PRSs derived from sufficiently large samples may be useful in the future for predicting conversion of transient tics to chronic tic disorders, as well as tic persistence and lifetime tic severity.

We present the case of a 12-year-old boy diagnosed with Tourette syndrome, who was successfully treated with a combination of vaporized medicinal cannabis and oral pure tetrahydrocannabinol (THC). Due to severe motor tics resulting in insomnia, the parents – both of whom were medical doctors – decided to initiate treatment with 0.02 g vaporized cannabis (Bedrocan with a THC content of 22% and a cannabidiol content of 1%; corresponding to a dose equivalent to 4.4 mg THC) without prior consultation of a Tourette expert. This treatment resulted – according to the parents’ report – in an immediate and nearly complete remission of the tics. Due to a further increase in tics, the parents therefore decided to implement a regular treatment with a combination of vaporized medicinal cannabis (up to 0.1 g cannabis per day, varieties Bedrocan and Amnesia Haze, corresponding to 22 mg THC/day) plus orally administered oil-based THC drops (maximum daily dose = 12.5 mg THC) resulting in a marked tic reduction. During a visit in our clinic, we were able to observe the reported beneficial effects 30 min after vaporization of 0.15 g cannabis (Amnesia Haze, equivalent to 33 mg THC; in addition, 7 mg oral THC were taken at home 6 h before the visit): tics, premonitory urges, and overall impairment significantly improved according to self-ratings, parent and clinician questionnaires. Importantly, no adverse events were reported. From this single case study, it is suggested that cannabis-based medicines and their combination (such as oral THC plus vaporized medicinal cannabis) are effective and safe in the treatment of severe tics in minors with TS. However, long-term follow-up is needed to confirm the beneficial treatment effects. We want to emphasize that in this boy treatment with cannabis was initiated by the parents before a consultation in our clinic has taken place. In our opinion, treatment with cannabis-based medicine in children should be regarded as a last-line treatment, when well-established treatments have failed to improve tics.

Efficient production of fuels and chemicals by metabolically engineered micro-organisms requires availability of precursor molecules for product pathways. In eukaryotic cell factories, heterologous product pathways are usually expressed in the cytosol, which may limit availability of precursors that are generated in other cellular compartments. In Saccharomyces cerevisiae, synthesis of the precursor molecule succinyl-Coenzyme A is confined to the mitochondrial matrix. To enable cytosolic synthesis of succinyl-CoA, we expressed the structural genes for all three subunits of the Escherichia coli α-ketoglutarate dehydrogenase (αKGDH) complex in S. cerevisiae. The E. coli lipoic-acid scavenging enzyme was co-expressed to enable cytosolic lipoylation of the αKGDH complex, which is required for its enzymatic activity. Size-exclusion chromatography and mass spectrometry indicated that the heterologously expressed αKGDH complex contained all subunits and that its size was the same as in E. coli. Functional expression of the heterologous complex was evident from increased αKGDH activity in the cytosolic fraction of yeast cell homogenates. In vivo cytosolic activity of the αKGDH complex was tested by constructing a reporter strain in which the essential metabolite 5-aminolevulinic acid could only be synthetized from cytosolic, and not mitochondrial, succinyl-CoA. To this end HEM1, which encodes the succinyl-CoA-converting mitochondrial enzyme 5-aminolevulinic acid (ALA) synthase, was deleted and a bacterial ALA synthase was expressed in the cytosol. In the resulting strain, complementation of ALA auxotrophy depended on activation of the αKGDH complex by lipoic acid addition. Functional expression of a bacterial αKGDH complex in yeast represents a vital step towards efficient yeast-based production of compounds such as 1,4-butanediol and 4-aminobutyrate, whose product pathways use succinyl-CoA as a precursor.

Genetic influences on psychiatric disorders transcend diagnostic boundaries, suggesting substantial pleiotropy of contributing loci. However, the nature and mechanisms of these pleiotropic effects remain unclear. We performed analyses of 232,964 cases and 494,162 controls from genome-wide studies of anorexia nervosa, attention-deficit/hyper-activity disorder, autism spectrum disorder, bipolar disorder, major depression, obsessive-compulsive disorder, schizophrenia, and Tourette syndrome. Genetic correlation analyses revealed a meaningful structure within the eight disorders, identifying three groups of inter-related disorders. Meta-analysis across these eight disorders detected 109 loci associated with at least two psychiatric disorders, including 23 loci with pleiotropic effects on four or more disorders and 11 loci with antagonistic effects on multiple disorders. The pleiotropic loci are located within genes that show heightened expression in the brain throughout the lifespan, beginning prenatally in the second trimester, and play prominent roles in neurodevelopmental processes. These findings have important implications for psychiatric nosology, drug development, and risk prediction.

Creatine metabolism is an important component of cellular energy homeostasis. Via the creatine kinase circuit, creatine derived from our diet or synthesized endogenously provides spatial and temporal maintenance of intracellular adenosine triphosphate (ATP) production; this is particularly important for cells with high or fluctuating energy demands. The use of this circuit by tissues within the female reproductive system, as well as the placenta and the developing fetus during pregnancy is apparent throughout the literature, with some studies linking perturbations in creatine metabolism to reduced fertility and poor pregnancy outcomes. Maternal dietary creatine supplementation during pregnancy as a safeguard against hypoxia-induced perinatal injury, particularly that of the brain, has also been widely studied in pre-clinical in vitro and small animal models. However, there is still no consensus on whether creatine is essential for successful reproduction. This review consolidates the available literature on creatine metabolism in female reproduction, pregnancy and the early neonatal period. Creatine metabolism is discussed in relation to cellular bioenergetics and de novo synthesis, as well as the potential to use dietary creatine in a reproductive setting. We highlight the apparent knowledge gaps and the research “road forward” to understand, and then utilize, creatine to improve reproductive health and perinatal outcomes.

Marek&rsquo, s disease (MD), caused by MD herpesvirus (MDV), is an economically important disease in chickens. The efficacy of the existing vaccines against evolving virulent stains may become limited and necessitates the development of novel antiviral strategies to protect poultry from MDV strains with increased virulence. The CRISPR/Cas9 system has emerged as a powerful genome editing tool providing an opportunity to develop antiviral strategies for the control of MDV infection. Here, we characterized Tol2 transposon constructs encoding Cas9 and guide RNAs (gRNAs) specific to the immediate early infected-cell polypeptide-4 (ICP4) of MDV. We generated transgenic chickens that constitutively express Cas9 and ICP4-gRNAs (gICP4) and challenged them via intraabdominal injection of MDV-1 Woodlands strain passage-19 (p19). Transgenic chickens expressing both gRNA/Cas9 had a significantly reduced replication of MDV in comparison to either transgenic Cas9-only or the wild-type (WT) chickens. We further confirmed that the designed gRNAs exhibited sequence-specific virus interference in transgenic chicken embryo fibroblast (CEF) expressing Cas9/gICP4 when infected with MDV but not with herpesvirus of turkeys (HVT). These results suggest that CRISPR/Cas9 can be used as an antiviral approach to control MDV infection in chickens, allowing HVT to be used as a vector for recombinant vaccines.

Introduction Preeclampsia is a serious complication affecting 5–8% of pregnancies. Central to its pathogenesis is placental hypoxia and inflammation which leads to secretion of soluble fms-like tyrosine kinase 1 (sFlt-1). sFlt-1 causes widespread endothelial dysfunction. The molecular mechanisms regulating sFlt-1 production remain poorly understood. Recently, a binding site for the nuclear factor activated T cells (NFAT) transcription factor has been found on fms-like tyrosine kinase 1 (FLT-1) promoter. Methods We assessed whether inhibiting NFAT impacts FLT-1, sFlt-1 and cytokine expression, as well as sFlt-1 secretion in primary cytotrophoblasts, placental explants and human umbilical vein endothelial cells (HUVECs). We investigated whether NFAT is regulated by hypoxia in primary cytotrophoblasts. We characterised the expression of NFAT1-4 in preterm preeclamptic compared to gestationally matched placentas. Results Inhibiting NFAT reduced FLT-1 and sFlt-1 splice variant e15a transcription, concordant with reduced total sFlt-1 and sFlt-1 e15a secretion from primary human cytotrophoblasts. This effect appeared tissue specific as inhibiting NFAT did not change sFlt-1 secretion from endothelial cells. Inhibiting NFAT also reduced transcription of inflammatory cytokines IL-1β and IL-10 in primary cytotrophoblasts. NFAT1 and NFAT3 mRNA expression were significantly increased under hypoxia (1% O2). Inhibiting NFAT under hypoxia significantly reduced FLT-1 and sFlt-1 e15a transcription, but did not reduce sFlt-1 secretion. NFAT mRNA and protein localisation was not different in preeclamptic compared to gestationally matched placenta. Discussion NFAT positively regulates placental FLT-1 and sFlt-1 e15a, secretion of sFlt-1 and inflammatory cytokine expression. It may be involved in the pathophysiology of preeclampsia.

In the past two decades, there has been increasing interest in the therapeutic potential of cannabis and single cannabinoids, mainly cannabidiol (CBD) and delta-9-tetrahydrocannabinol (THC). THC and cannabis products rich in THC exert their effects mainly through the activation of cannabinoid receptors (CB1 and CB2). Since 1975, 140 controlled clinical trials using different cannabinoids or whole-plant preparations for the treatment of a large number of disorders and symptoms have been conducted. Results have led to the approval of cannabis-based medicines [dronabinol, nabilone, and the cannabis extract nabiximols (Sativex®, THC:CBD = 1:1)] as well as cannabis flowers in several countries. Controlled clinical studies provide substantial evidence for the use of cannabinoid receptor agonists in cancer chemotherapy induced nausea and vomiting, appetite loss and cachexia in cancer and HIV patients, neuropathic and chronic pain, and in spasticity in multiple sclerosis. In addition, there is also some e...

Muscle weakness in MuSK myasthenia gravis (MG) is caused predominantly by IgG4 antibodies which block MuSK signalling and destabilize neuromuscular junctions. We determined whether the binding pattern of MuSK IgG4 antibodies change throughout the disease course ("epitope spreading"), and affect disease severity or treatment responsiveness.We mapped the MuSK epitopes of 255 longitudinal serum samples of 53 unique MuSK MG patients from three independent cohorts with ELISA.Antibodies against the MuSK Iglike-1 domain determine disease severity. Epitope spreading outside this domain did not contribute to disease severity nor to pyridostigmine responsiveness. This provides a rationale for epitope specific treatment strategies. (C) 2016 Elsevier B.V. All rights reserved.

Resistant tumours are thought to arise from the action of Darwinian selection on genetically heterogenous cancer cell populations. However, simple clonal selection is inadequate to describe the late relapses often characterising luminal breast cancers treated with endocrine therapy (ET), suggesting a more complex interplay between genetic and non-genetic factors. Partially, this is due to our limited understanding on the effect of ET at the single cell level. In the present study, we dissect the contributions of clonal genetic diversity and transcriptional plasticity during the early and late phases of ET at single-cell resolution. Using single-cell RNA-sequencing and imaging we disentangle the transcriptional variability of plastic cells and define a rare sub-population of pre-adapted (PA) cells which undergoes further transcriptomic reprogramming and copy number changes to acquire full resistance. PA cells show reduced oestrogen receptor α activity but increased features of quiescence and migration. We find evidence for sub-clonal expression of this PA signature in primary tumours and for dominant expression in clustered circulating tumour cells. We propose a multi-step model for ET resistance development and advocate the use of stage-specific biomarkers.

Acetone monooxygenase (ACMO) is a unique member of the Baeyer–Villiger monooxygenase (BVMO) family based on its ability to act on small ketones, such as acetone. Herein, we performed a kinetic analysis of ACMO from the propane-utilizing bacterium Gordonia sp. strain TY-5 to assess its preference for smaller ketone substrates. Steady state kinetic analysis of ACMO with a range of linear (C3–C7) and cyclic ketones (C4–C6) reveals that the enzyme elicits the highest catalytic efficiency towards butanone and cyclobutanone. Stopped-flow and inhibition studies further revealed that ACMO has a relatively weak binding affinity for the coenzyme with a dissociation constant of 120 μM. We show through mutagenesis that sequence variation in the residue that coordinates to the 2′-phosphate of NADP(H) partially accounts for the weaker binding affinity observed. As for shown for related BVMOs, NADP+ stabilizes the C4a-peroxyflavin intermediate in ACMO; however, the rate of its formation is considerably slower in ACMO. The observed rate constant for NADPH-dependent flavin reduction was 60 s−1 at 25 °C, and experiments performed with 4(R)-[4-2H]NADPH confirm that the C4-pro-R-hydride from the nicotinamide ring is transferred to the FAD. The latter experimental result suggests that the nicotinamide ring rotates within the active site to carry out its two functional roles: reduction of the FAD cofactor and stabilization of the C4a-peroxyflavin adduct. Electronic supplementary material The online version of this article (10.1186/s13568-018-0709-x) contains supplementary material, which is available to authorized users.

Cyclohexanone monooxygenase (CHMO) uses NADPH and O 2 to insert oxygen into an array of (a)cyclic ketones to form esters or lactones. Herein, the role of two conserved active site residues (R327 and D57) in controlling the binding mode of NADP(H) was investigated. Wild type CHMO elicits a kinetic isotope effect (KIE) of 4.7 ± 0.1 and 1.1 ± 0.1 with 4( R )-[4- 2 H]NADPH and 4( S )-[4- 2 H]NADPH, respectively, consistent with transfer of the pro R hydrogen to FAD. Strikingly, the R327K variant appears to lack stereospecificity for hydride transfer as a KIE of 1.5 ± 0.1 and 2.5 ± 0.1 was observed for the pro R and pro S deuterated forms of NADPH. 1 H NMR of the NADP + products confirmed that the R327K variant abstracts either the pro R or pro S hydrogen from NADPH. While the D57A variant retained stereospecificity for the pro R hydrogen, this substitution resulted in slow decomposition of the C4a-peroxyflavin intermediate in the presence of cyclohexanone. Based on published structures of a related flavin monooxygenase, we suggest that elimination of the hydrogen bond between D57 and R327 in the D57A variant causes R327 to adopt a substrate-induced conformation that slows substrate access to the active site, thereby prolonging the lifetime of the C4a-peroxyflavin intermediate.

Pluripotent stem cells (PSCs) can self-renew indefinitely while maintaining the ability to generate all cell types of the body. This plasticity is proposed to require heterogeneity in gene expression, driving a metastable state which may allow flexible cell fate choices. Contrary to this, naive PSC grown in fully defined ‘2i’ environmental conditions, containing small molecule inhibitors of MEK and GSK3 kinases, show homogenous pluripotency and lineage marker expression. However, here we show that 2i induces greater genome-wide heterogeneity than traditional serum-containing growth environments at the population level across both male and female PSCs. This heterogeneity is dynamic and reversible over time, consistent with a dynamic metastable equilibrium of the pluripotent state. We further show that the 2i environment causes increased heterogeneity in the calcium signalling pathway at both the population and single-cell level. Mechanistically, we identify loss of robustness regulators in the form of negative feedback to the upstream EGF receptor. Our findings advance the current understanding of the plastic nature of the pluripotent state and highlight the role of signalling pathways in the control of transcriptional heterogeneity. Furthermore, our results have critical implications for the current use of kinase inhibitors in the clinic, where inducing heterogeneity may increase the risk of cancer metastasis and drug resistance.

Zoonotic and foodborne diseases pose a significant burden, decreasing both human and animal health. Modifying chickens to overexpress antimicrobials has the potential to decrease bacterial growth on poultry products and boost chicken innate immunity. Chickens overexpressing either ovotransferrin or avian β-defensin-3 (AvβD3) were generated using Tol-2 transposons. Transgene expression at the RNA and protein level was seen in egg white, breast muscle, and serum. There were significant differences in the immune cell populations in the blood, bursa, and spleen associated with transgene expression including an increased proportion of CD8+ cells in the blood of ovotransferrin and AvβD3 transgenic birds. Expression of the antimicrobials inhibited the in vitro growth of human and chicken bacterial pathogens and spoilage bacteria. For example, transgene expression significantly reduced growth of aerobic and coliform bacteria in breast muscle and decreased the growth of Salmonella enterica in egg white. Overall these results indicate that overexpression of antimicrobials in the chicken can impact the immune system and increase the antimicrobial capacity of poultry products.

Baeyer-Villiger monooxygenases are flavoenzymes that use NADPH and O2 to convert ketones to esters or lactones. A diagnostic feature of BVMO catalysis is the dual role of the pyridine nucleotide: NADPH functions as a reductant of the FAD cofactor and the resulting NADP+ acts to stabilize the ensuing C4a-peroxyflavin intermediate. Using cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 as a model system, we investigated the catalytic role of T187 and W490, which hydrogen bond to the phosphate and ribose of the nicotinamide mononucleotide half of NADP(H), respectively. Eliminating either hydrogen bond through creation of a T187A or a W490F variant leads to a 15-fold reduction in turnover of cyclohexanone. Substitution of either residue does not affect the rate of FAD reduction or the coupling efficiency. Rather, T187A and W490F disrupt distinct steps of the oxidative half-reaction. Kinetic and spectroscopic analysis of T187A reveals that this residue is critical for locking NADP+ in a configuration that dramatically accelerates O2 activation by the reduced flavin. W490 also promotes O2 activation (albeit less so than T187) and accelerates the reaction between the C4a-peroxyflavin and cyclohexanone. The results provide insight into the conformation of CHMO and the coenzyme for optimal catalysis.