Recapitulation of Skewed X-Inactivation in Female Ornithine Transcarbamylase-Deficient Primary Human Hepatocytes in the FRG Mouse: A Novel System for Developing Epigenetic Therapies.

Realization of the immense therapeutic potential of epigenetic editing requires development of clinically predictive model systems that faithfully recapitulate relevant aspects of the target disease pathophysiology. In female patients with ornithine transcarbamylase (OTC) deficiency, an X-linked condition, skewed inactivation of the X chromosome carrying the wild-type OTC allele is associated with increased disease severity. The majority of affected female patients can be managed medically, but a proportion require liver transplantation. With rapid development of epigenetic editing technology, reactivation of silenced wild-type OTC alleles is becoming an increasingly plausible therapeutic approach. Toward this end, privileged access to explanted diseased livers from two affected female infants provided the opportunity to explore whether engraftment and expansion of dissociated patient-derived hepatocytes in the FRG mouse might produce a relevant model for evaluation of epigenetic interventions. Hepatocytes from both infants were successfully used to generate chimeric mouse-human livers, in which clusters of primary human hepatocytes were either OTC positive or negative by immunohistochemistry (IHC), consistent with clonal expansion from individual hepatocytes in which the mutant or wild-type OTC allele was inactivated, respectively. Enumeration of the proportion of OTC-positive or -negative human hepatocyte clusters was consistent with dramatic skewing in one infant and minimal to modest skewing in the other. Importantly, IHC and fluorescence-activated cell sorting analysis of intact and dissociated liver samples from both infants showed qualitatively similar patterns, confirming that the chimeric mouse-human liver model recapitulated the native state in each infant. Also of importance was the induction of a treatable metabolic phenotype, orotic aciduria, in mice, which correlated with the presence of clonally expanded OTC-negative primary human hepatocytes. We are currently using this unique model to explore CRISPR-dCas9-based epigenetic targeting strategies in combination with efficient adeno-associated virus (AAV) gene delivery to reactivate the silenced functional OTC gene on the inactive X chromosome.

The P450 multigene family of Fontainea and insights into diterpenoid synthesis.

BACKGROUND: Cytochrome P450s (P450s) are enzymes that play critical roles in the biosynthesis of physiologically important compounds across all organisms. Although they have been characterised in a large number of plant species, no information relating to these enzymes are available from the genus Fontainea (family Euphorbiaceae). Fontainea is significant as the genus includes species that produce medicinally significant epoxy-tigliane natural products, one of which has been approved as an anti-cancer therapeutic. RESULTS: A comparative species leaf metabolome analysis showed that Fontainea species possess a chemical profile different from various other plant species. The diversity and expression profiles of Fontainea P450s were investigated from leaf and root tissue. A total of 103 and 123 full-length P450 genes in Fontainea picrosperma and Fontainea venosa, respectively (and a further 127/125 partial-length) that were phylogenetically classified into clans, families and subfamilies. The majority of P450 identified are most active within root tissue (66.2% F. picrosperma, 65.0% F. venosa). Representatives within the CYP71D and CYP726A were identified in Fontainea that are excellent candidates for diterpenoid synthesis, of which CYP726A1, CYP726A2 and CYP71D1 appear to be exclusive to Fontainea species and were significantly more highly expressed in root tissue compared to leaf tissue. CONCLUSION: This study presents a comprehensive overview of the P450 gene family in Fontainea that may provide important insights into the biosynthesis of the medicinally significant epoxy-tigliane diterpenes found within the genus.

Rituximab impedes natural killer cell function in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis patients: A pilot in vitro investigation.

BACKGROUND: A recent in vitro pilot investigation reported Rituximab significantly reduced natural killer (NK) cell cytotoxicity in healthy donors. Chronic fatigue syndrome/Myalgic encephalomyelitis (CFS/ME) is a debilitating disorder of unknown etiology. A consistent finding is a significant reduction in NK cell cytotoxicity. Rituximab has been reported having questionable potential therapeutic benefits for the treatment of CFS/ME, however, the potential effects of Rituximab on NK cell cytotoxicity in CFS/ME patients are yet to be determined. METHODS: A total of eight CFS/ME patients (48.63 ± 15.69 years) and nine non-fatigued controls (NFC) (37.56 ± 11.06 years) were included using the Fukuda case definition. Apoptotic function, lytic proteins and degranulation markers were measured on isolated NK cells using flow cytometry following overnight incubation with Rituximab at 10 µg/ml and 100 µg/ml. RESULTS: There was a significant reduction in NK cell lysis between CFS/ME patients and NFC following incubation with Rituximab at 100 µg/ml at 12.5:1 and 6.25:1 effecter-target (E:T) ratios (p < 0.05). However, there was no significant difference for NFC following incubation with Rituximab at 10 µg/ml and 100 µg/ml. There was no significant difference between CFS/ME patients and NFC for granzyme A and granzyme B prior to incubation with Rituximab and following overnight incubation with Rituximab at 10 µg/ml. There was a significant decrease in granzyme B in CFS/ME patients compared to NFC with 100 µg/ml of Rituximab prior to K562 cells stimulation (p < 0.05). There was a significant increase in CD107a (p < 0.05) and CD107b expression (p < 0.01) in NFC after stimulation with K562 cells prior to incubation with Rituximab. There was a significant increase in CD107b expression between CFS/ME patients and NFC prior to incubation with Rituximab and without stimulation of K562 cells (p < 0.01). Importantly, there was a significant increase in CD107b following overnight incubation with 100 µg/ml of Rituximab in NFC prior to K562 cells stimulation (p < 0.01). CONCLUSION: This study reports significant decreases in NK cell lysis and a significant increase in NK cell degranulation following Rituximab incubation in vitro in CFS/ME patients, suggesting Rituximab may be toxic for NK cells. Caution should be observed in clinical trials until further investigations in a safe and controlled in vitro setting are completed.

A targeted genome association study examining transient receptor potential ion channels, acetylcholine receptors, and adrenergic receptors in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis.

BACKGROUND: Chronic Fatigue Syndrome, also known as Myalgic Encephalomyelitis (CFS/ME) is a debilitating condition of unknown aetiology. It is characterized by a range of physiological effects including neurological, sensory and motor disturbances. This study examined candidate genes for the above clinical manifestations to identify single nucleotide polymorphism (SNP) alleles associated with CFS/ME compared with healthy controls. METHODS: DNA was extracted and whole genome genotyping was performed using the HumanOmniExpress BeadChip array. Gene families for transient receptor potential ion channels, acetylcholine receptors, and adrenergic receptors, and acetylcholinesterase were targeted. The frequency of each SNP and their association between CFS/ME and healthy controls was examined using Fisher's exact test, and to adjust for multiple testing, False Detection Rate (FDR) and Bonferroni corrections were applied (p < 0.05). RESULTS: The study included 172 participants, consisting of 95 Fukuda defined CFS/ME patients (45.8 ± 8.9; 69 % female) and 77 healthy controls (42.3 ± 10.3; 63 % female). A total of 950 SNPs were included for analysis. 60 significant SNPs were associated with CFS/ME compared with healthy controls. After applying FDR and Bonferroni corrections, SNP rs2322333 in adrenergic receptor α1 (ADRA1A) was higher in CFS/ME compared with healthy controls (45.3 % vs. 23.4 %; p = 0.059). The genotype class that was homozygous minor (AA) was substantially lower in CFS/ME compared with healthy controls (4.2 % vs. 24.7 %). CONCLUSIONS: This study reports for the first time the identification of ADRA1A and a possible association between CFS/ME and genotype classes. Further examination of the functional role of this class of adrenergic receptors may elucidate the cause of particular clinical manifestations observed in CFS/ME.

Attitudes in the general population towards hemi-craniectomy for middle cerebral artery (MCA) infarction. A population-based survey.

BACKGROUND: Decompressive hemicraniectomy reduces mortality after space-occupying MCA infarction. Data on the general public's opinion toward interventions that can save lives but leave the survivors impaired are lacking. METHODS: In this population-based epidemiological study in a German city, we surveyed 312 adults in a telephone interview. Here, we presented a scenario of a space-occupying MCA infarct. We evaluated probands' attitude toward decompressive surgery in general, and toward outcome scenarios according to Rankin scale (RS) definitions. RESULTS: 312 persons (157 women, 52 ± 20 years) were interviewed. 58 persons had difficulty comprehending the proposed scenario, most of them being of advanced age (79 ± 5 years). From the remaining 254 responders 5 (2%) persons favoured surgical intervention, 149 (58%) were undecided, and 100 (39%) were opposed to surgery. The number of individuals opting for surgery rose in scenarios with a better outcome: If very severe impairment was anticipated (RS 5), only 3 (1%) persons favored surgery. With severe (RS 4), moderate (RS3), and slight impairment (RS2) the numbers were at 16 (6%), 60 (24%), and 161 (63%), respectively. We found no association with age, sex, religion, education, self-estimated health status, or marital status. CONCLUSIONS: Explaining complex medical situations to laypersons poses a major problem, particularly to those of old age. Only a minority favors life-saving medical interventions if survival is associated with deficits of unpredictable degree. The majority of persons does not favor intervention even if only moderate impairment is anticipated. Decompressive surgery may in fact be against the values of many individuals.