De novo coding variants in the AGO1 gene cause a neurodevelopmental disorder with intellectual disability.

BACKGROUND: High-impact pathogenic variants in more than a thousand genes are involved in Mendelian forms of neurodevelopmental disorders (NDD). METHODS: This study describes the molecular and clinical characterisation of 28 probands with NDD harbouring heterozygous AGO1 coding variants, occurring de novo for all those whose transmission could have been verified (26/28). RESULTS: A total of 15 unique variants leading to amino acid changes or deletions were identified: 12 missense variants, two in-frame deletions of one codon, and one canonical splice variant leading to a deletion of two amino acid residues. Recurrently identified variants were present in several unrelated individuals: p.(Phe180del), p.(Leu190Pro), p.(Leu190Arg), p.(Gly199Ser), p.(Val254Ile) and p.(Glu376del). AGO1 encodes the Argonaute 1 protein, which functions in gene-silencing pathways mediated by small non-coding RNAs. Three-dimensional protein structure predictions suggest that these variants might alter the flexibility of the AGO1 linker domains, which likely would impair its function in mRNA processing. Affected individuals present with intellectual disability of varying severity, as well as speech and motor delay, autistic behaviour and additional behavioural manifestations. CONCLUSION: Our study establishes that de novo coding variants in AGO1 are involved in a novel monogenic form of NDD, highly similar to the recently reported AGO2-related NDD.

In vitro and ex vivo experimental models for evaluation of intranasal systemic drug delivery as well as direct nose-to-brain drug delivery.

The intranasal route of administration provides a noninvasive method to deliver drugs into the systemic circulation and/or directly into the brain. Direct nose-to-brain drug delivery offers the possibility to treat central nervous system diseases more effectively, as it can evade the blood-brain barrier. In vitro and ex vivo intranasal models provide a means to investigate physiological and pharmaceutical factors that could play a role in drug delivery across the nasal epithelium as well as to determine the mechanisms involved in drug absorption from the nose. The development and implementation of cost-effective pharmacokinetic models for intranasal drug delivery with good in vitro-in vivo correlation can accelerate pharmaceutical drug product development and improve economic and ecological aspects by reducing the time and costs spent on animal studies. Special considerations should be made with regard to the purpose of the in vitro/ex vivo study, namely, whether it is intended to predict systemic or brain delivery, source and site of tissue or cell sampling, viability window of selected model, and the experimental setup of diffusion chambers. The type of model implemented should suit the relevant needs and requirements of the project, researcher, and interlaboratory. This review aims to provide an overview of in vitro and ex vivo models that have been developed to study intranasal and direct nose-to-brain drug delivery.

Geographical and seasonal phytochemical variation of Artemisia afra Jacq. ex Willd.

INTRODUCTION: Artemisia afra Jacq. ex. Willd. (Asteraceae) is a popular traditional medicine in South Africa, mainly used in the form of an infusion, for the treatment of respiratory ailments. Quality control methods are limited and phytochemical variation for the infusion is not well known. OBJECTIVE: To develop a sensitive quality control method for A. afra infusions by validating a liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS) method and quantitatively comparing six marker compounds in A. afra samples collected from different locations and over a 12-month period. MATERIAL AND METHODS: There was a multiple reaction monitoring method optimised and validated, according to ICH and FDA guidelines, to quantify the chemical markers present in infusions. RESULTS: The chemistry differed significantly and interestingly, with an interchangeable trend between chlorogenic acid (CGA) and 4,5-dicaffeoylquinic acid (DCQA) observed in the samples collected monthly, elevated levels of CGA during winter and elevated levels of DCQA during summer. The remaining four markers showed a steady decrease as winter approached and a steady increase as summer approached. The ranges of the six markers were the following: CGA (0.68-14.68 µg/mg), DCQA (0.005-8.110 µg/mg), quercetin (0.01-0.65 µg/mg), luteolin (0.05-1.30 ng/mg), scopoletin (0.10-1.14 µg/mg), scopolin (0.03-1.21 µg/mg). CONCLUSIONS: A sensitive LC-ESI-MS/MS method was developed, validated, and used to quantify six marker compounds. The results indicated a large degree of phytochemical variation occurred across all samples tested, which highlights the importance of producing herbal medicine under controlled conditions and the necessity of analytical quality control methods.

Lessons learned from 40 novel PIGA patients and a review of the literature.

OBJECTIVE: To define the phenotypic spectrum of phosphatidylinositol glycan class A protein (PIGA)-related congenital disorder of glycosylation (PIGA-CDG) and evaluate genotype-phenotype correlations. METHODS: Our cohort encompasses 40 affected males with a pathogenic PIGA variant. We performed a detailed phenotypic assessment, and in addition, we reviewed the available clinical data of 36 previously published cases and assessed the variant pathogenicity using bioinformatical approaches. RESULTS: Most individuals had hypotonia, moderate to profound global developmental delay, and intractable seizures. We found that PIGA-CDG spans from a pure neurological phenotype at the mild end to a Fryns syndrome-like phenotype. We found a high frequency of cardiac anomalies including structural anomalies and cardiomyopathy, and a high frequency of spontaneous death, especially in childhood. Comparative bioinformatical analysis of common variants, found in the healthy population, and pathogenic variants, identified in affected individuals, revealed a profound physiochemical dissimilarity of the substituted amino acids in variant constrained regions of the protein. SIGNIFICANCE: Our comprehensive analysis of the largest cohort of published and novel PIGA patients broadens the spectrum of PIGA-CDG. Our genotype-phenotype correlation facilitates the estimation on pathogenicity of variants with unknown clinical significance and prognosis for individuals with pathogenic variants in PIGA.

Estimating the effect size of the 15Q11.2 BP1-BP2 deletion and its contribution to neurodevelopmental symptoms: recommendations for practice.

BACKGROUND: The 15q11.2 deletion is frequently identified in the neurodevelopmental clinic. Case-control studies have associated the 15q11.2 deletion with neurodevelopmental disorders, and clinical case series have attempted to delineate a microdeletion syndrome with considerable phenotypic variability. The literature on this deletion is extensive and confusing, which is a challenge for genetic counselling. The aim of this study was to estimate the effect size of the 15q11.2 deletion and quantify its contribution to neurodevelopmental disorders. METHODS: We performed meta-analyses on new and previously published case-control studies and used statistical models trained in unselected populations with cognitive assessments. We used new (n=241) and previously published (n=150) data from a clinically referred group of deletion carriers. 15q11.2 duplications (new n=179 and previously published n=35) were used as a neutral control variant. RESULTS: The deletion decreases IQ by 4.3 points. The estimated ORs and respective frequencies in deletion carriers for intellectual disabilities, schizophrenia and epilepsy are 1.7 (3.4%), 1.5 (2%) and 3.1 (2.1%), respectively. There is no increased risk for heart malformations and autism. In the clinically referred group, the frequency and nature of symptoms in deletions are not different from those observed in carriers of the 15q11.2 duplication suggesting that most of the reported symptoms are due to ascertainment bias. CONCLUSIONS: We recommend that the deletion should be classified as 'pathogenic of mild effect size'. Since it explains only a small proportion of the phenotypic variance in carriers, it is not worth discussing in the developmental clinic or in a prenatal setting.

Genetic variants in the KDM6B gene are associated with neurodevelopmental delays and dysmorphic features.

Lysine-specific demethylase 6B (KDM6B) demethylates trimethylated lysine-27 on histone H3. The methylation and demethylation of histone proteins affects gene expression during development. Pathogenic alterations in histone lysine methylation and demethylation genes have been associated with multiple neurodevelopmental disorders. We have identified a number of de novo alterations in the KDM6B gene via whole exome sequencing (WES) in a cohort of 12 unrelated patients with developmental delay, intellectual disability, dysmorphic facial features, and other clinical findings. Our findings will allow for further investigation in to the role of the KDM6B gene in human neurodevelopmental disorders.

Diagnostic implications of genetic copy number variation in epilepsy plus.

OBJECTIVE: Copy number variations (CNVs) represent a significant genetic risk for several neurodevelopmental disorders including epilepsy. As knowledge increases, reanalysis of existing data is essential. Reliable estimates of the contribution of CNVs to epilepsies from sizeable populations are not available. METHODS: We assembled a cohort of 1255 patients with preexisting array comparative genomic hybridization or single nucleotide polymorphism array based CNV data. All patients had "epilepsy plus," defined as epilepsy with comorbid features, including intellectual disability, psychiatric symptoms, and other neurological and nonneurological features. CNV classification was conducted using a systematic filtering workflow adapted to epilepsy. RESULTS: Of 1097 patients remaining after genetic data quality control, 120 individuals (10.9%) carried at least one autosomal CNV classified as pathogenic; 19 individuals (1.7%) carried at least one autosomal CNV classified as possibly pathogenic. Eleven patients (1%) carried more than one (possibly) pathogenic CNV. We identified CNVs covering recently reported (HNRNPU) or emerging (RORB) epilepsy genes, and further delineated the phenotype associated with mutations of these genes. Additional novel epilepsy candidate genes emerge from our study. Comparing phenotypic features of pathogenic CNV carriers to those of noncarriers of pathogenic CNVs, we show that patients with nonneurological comorbidities, especially dysmorphism, were more likely to carry pathogenic CNVs (odds ratio = 4.09, confidence interval = 2.51-6.68; P = 2.34 × 10-9 ). Meta-analysis including data from published control groups showed that the presence or absence of epilepsy did not affect the detected frequency of CNVs. SIGNIFICANCE: The use of a specifically adapted workflow enabled identification of pathogenic autosomal CNVs in 10.9% of patients with epilepsy plus, which rose to 12.7% when we also considered possibly pathogenic CNVs. Our data indicate that epilepsy with comorbid features should be considered an indication for patients to be selected for a diagnostic algorithm including CNV detection. Collaborative large-scale CNV reanalysis leads to novel declaration of pathogenicity in unexplained cases and can promote discovery of promising candidate epilepsy genes.

Clinical delineation of the PACS1-related syndrome--Report on 19 patients.

We report on 19 individuals with a recurrent de novo c.607C>T mutation in PACS1. This specific mutation gives rise to a recognizable intellectual disability syndrome. There is a distinctive facial appearance (19/19), characterized by full and arched eyebrows, hypertelorism with downslanting palpebral fissures, long eye lashes, ptosis, low set and simple ears, bulbous nasal tip, wide mouth with downturned corners and a thin upper lip with an unusual "wavy" profile, flat philtrum, and diastema of the teeth. Intellectual disability, ranging from mild to moderate, was present in all. Hypotonia is common in infancy (8/19). Seizures are frequent (12/19) and respond well to anticonvulsive medication. Structural malformations are common, including heart (10/19), brain (12/16), eye (10/19), kidney (3/19), and cryptorchidism (6/12 males). Feeding dysfunction is presenting in infancy with failure to thrive (5/19), gastroesophageal reflux (6/19), and gastrostomy tube placement (4/19). There is persistence of oral motor dysfunction. We provide suggestions for clinical work-up and management and hope that the present study will facilitate clinical recognition of further cases.

Mutation of the iron-sulfur cluster assembly gene IBA57 causes severe myopathy and encephalopathy.

Two siblings from consanguineous parents died perinatally with a condition characterized by generalized hypotonia, respiratory insufficiency, arthrogryposis, microcephaly, congenital brain malformations and hyperglycinemia. Catalytic activities of the mitochondrial respiratory complexes I and II were deficient in skeletal muscle, a finding suggestive of an inborn error in mitochondrial biogenesis. Homozygosity mapping identified IBA57 located in the largest homozygous region on chromosome 1 as a culprit candidate gene. IBA57 is known to be involved in the biosynthesis of mitochondrial [4Fe-4S] proteins. Sequence analysis of IBA57 revealed the homozygous mutation c.941A > C, p.Gln314Pro. Severely decreased amounts of IBA57 protein were observed in skeletal muscle and cultured skin fibroblasts from the affected subjects. HeLa cells depleted of IBA57 showed biochemical defects resembling the ones found in patient-derived cells, including a decrease in various mitochondrial [4Fe-4S] proteins and in proteins covalently linked to lipoic acid (LA), a cofactor produced by the [4Fe-4S] protein LA synthase. The defects could be complemented by wild-type IBA57 and partially by mutant IBA57. As a result of the mutation, IBA57 protein was excessively degraded, an effect ameliorated by protease inhibitors. Hence, we propose that the mutation leads to partial functional impairment of IBA57, yet the major pathogenic impact is due to its proteolytic degradation below physiologically critical levels. In conclusion, the ensuing lethal complex biochemical phenotype of a novel metabolic syndrome results from multiple Fe/S protein defects caused by a deficiency in the Fe/S cluster assembly protein IBA57.

Effect of acute stresses on zebra fish (Danio rerio) metabolome measured by NMR-based metabolomics.

We applied an acute stress model to zebra fish in order to measure the changes in the metabolome due to biological stress. This was done by submitting the fish to fifteen minutes of acute confinement (netting) stress, and then five minutes for the open field and light/dark field tests. A polar extract of the zebra fish was then subjected to (1)H nuclear magnetic spectroscopy. Multivariate data analysis of the spectra showed a clear separation associated to a wide range of metabolites between zebra fish that were submitted to open field and light/dark field tests. Alanine, taurine, adenosine, creatine, lactate, and histidine were high in zebra fish to which the light/dark field test was applied, regardless of stress, while acetate and isoleucine/lipids appeared to be higher in zebra fish exposed to the open field test. These results show that any change in the environment, even for a small period of time, has a noticeable physiological impact. This research provides an insight of how different mechanisms are activated under different environments to maintain the homeostasis of the body. It should also contribute to establish zebra fish as a model for metabolomics studies.

Is artemisinin the only antiplasmodial compound in the Artemisia annua tea infusion? An in vitro study.

In our ongoing investigation into Artemisia annua for the treatment of malaria, we decided to study the possibility that synergism might enhance the efficacy of artemisinin. Our main objective was to test tea infusions and nonpolar extracts prepared from different A. annua varieties against Plasmodium falciparum in vitro in order to determine if synergism will increase the effectiveness of artemisinin in the samples as compared to pure artemisinin. We found that the IC50 of artemisinin in the tea and nonpolar extracts was not significantly different to the IC50 of pure artemisinin. We could show that the year and country of harvest or storage conditions did not have any influence on the activity and that it narrowly followed the concentration of artemisinin in all the extracts. In conclusion, based on these in vitro results, artemisinin seems to be the only active antiplasmodial compound in A. annua.

Phytochemistry of the carnivorous sundew genus Drosera (Droseraceae) - future perspectives and ethnopharmacological relevance.

Species of the carnivorous genus Drosera L. have long been a source of valuable natural products. The various phytochemicals characteristic of these species, particularly 1,4-naphthoquinones and flavonoids, have contributed to the diverse utilization of sundews in traditional medicine systems worldwide. A growing number of studies have sought to investigate the comparative phytochemistry of Drosera species for improved sources of pharmaceutically important compounds. The outcomes of these studies are here collated, with emergent trends discussed in detail. Important factors which affect production of secondary metabolites in plants are critically examined, such as environmental influences and in vitro culture, and recommendations subsequently presented based on this. Explicitly, the current review aims to i) present an updated, comprehensive listing of the phytochemical constituents of the genus (including quantitative data where available), ii) summarize important factors which may influence the production of phytopharmaceuticals in plants, and iii) recommend guidelines for future research based on the above, including improved standardization and quality control. We have also included a section discussing future perspectives of research on Drosera spp. based on three different research lines i) the potential to produce much needed lead compounds for treatment of tuberculosis, ii) the potential role of anthocyanins in nitrogen transport, and iii) research into 'Natural Deep Eutectic' solvents produced by Drosera spp. in the droplets or 'dew' employed to capture insect prey.

The use of bio-guided fractionation to explore the use of leftover biomass in Dutch flower bulb production as allelochemicals against weeds.

A major problem in flower bulb cultivation is weed control. Synthetic herbicides are mainly used, although they cause a range of problems, and integrated weed control through application of naturally occurring allelochemicals would be highly desirable. Flower bulb production creates large amounts of leftover biomass. Utilizing this source for weed control may provide new applications of the bulb crops. We therefore screened 33 flower bulb extracts for allelochemical activity against weeds. Several methanol and chloroform extracts were observed to inhibit germination and growth of Senecio vulgaris L. and Lolium perenne L., as representatives of di- and mono-cotyledonous weeds, respectively. Narciclasine was identified as the bioactive compound in Narcissus. The extract of Amaryllis belladonna L. was equally active, but did not contain any narciclasine. Bioassay-guided fractionation of the A. belladonna extract resulted in the identification of lycorine as the bio-active compound. The IC50 measured for radicle growth inhibition was 0.10 µM for narciclasine and 0.93 µM for lycorine, compared to 0.11 mM of chlorpropham, a synthetic herbicide. Therefore, the leftover biomass from the spring bulb industry represents an interesting potential source for promising allelochemicals for further studies on weed growth inhibition.

Influence of Drought and Heat Stress on Mineral Content, Antioxidant Activity and Bioactive Compound Accumulation in Four African Amaranthus Species.

Drought and heat stress is known to influence the accumulation of mineral content, antioxidant activity, phenolics, flavonoids and other bioactive compounds in many tolerant leafy vegetables. Amaranthus plants can tolerate adverse weather conditions, especially drought and heat. Therefore, evaluating the influence of drought and heat stress on commercially and medically important crop species like Amaranthus is important to grow the crop for optimal nutritional and medicinal properties. This study investigated the influence of drought and heat stress and a combination of both on the accumulation of phenolic and flavonoid compounds and the antioxidant capacity of African Amaranthus caudatus, A. hypochondriacus, A. cruentus and A. spinosus. Phenolic and flavonoid compounds were extracted with methanol and aqueous solvents and were quantified using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Caffeic acid was the main phenolic compound identified in aqueous extracts of A. caudatus and A. hypochondriacus. Rutin was the most abundant flavonoid compound in all the Amaranthus species tested, with the highest concentration found in A. caudatus. The results suggest a strong positive, but species and compound-specific effect of drought and heat stress on bioactive compounds accumulation. We concluded that heat stress at 40 °C under well-watered conditions and combined drought and heat stress (at 30 °C and 35 °C) appeared to induce the accumulation of caffeic acid and rutin. Hence, cultivation of these species in semi-arid and arid areas is feasible.

Development of a HPLC Method for Analysis of a Combination of Clofazimine, Isoniazid, Pyrazinamide, and Rifampicin Incorporated into a Dermal Self-Double-Emulsifying Drug Delivery System.

We describe the development and validation of a new high performance liquid chromatography (HPLC) method for analysis of a combination of the first-line anti-tubercular drugs isoniazid, pyrazinamide, and rifampicin together with clofazimine. This is a unique challenge since clofazimine and rifampicin are relatively highly lipophilic drugs, whereas isoniazid and pyrazinamide are considerably more hydrophilic. Thus, clear separation of peaks and quantification of four individual drugs can present difficulties during the development of an analytical method. Detection was established at two wavelengths-254 nm for isoniazid and pyrazinamide and 320 nm for clofazimine and rifampicin. Gradient elution was employed using 0.1% aqueous formic acid (A) and acetonitrile (B); clear separation of the four drugs was achieved within 10 min. A linear relationship was indicated by a correlation coefficient (r2) of 0.9999 for each anti-tubercular drug, respectively. The limit of detection (LOD) for the individual drugs was 0.70 µg/mL (isoniazid), 0.30 µg/mL (pyrazinamide), 0.20 µg/mL (rifampicin) and 0.20 µg/mL (clofazimine). Precision experiments rendered a mean recovery percentage of 101.25% (isoniazid), 98.70% (pyrazinamide), 99.68% (rifampicin) and 97.14% (clofazimine). This HPLC method was validated and is reliable, repeatable, and accurate for the purpose of conducting simultaneous HPLC analyses of the four anti-tubercular drugs.

The effect of environmental stressors on the anticancer potential of Amaranthus hypochondriacus aqueous extracts and fractions.

Some Amaranthus species have been shown to have pharmacological properties such as activity against cancer, and it is also used as a traditional herbal medicine in many rural parts of the world. The (3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide assay was used as a screening tool to determine the approximate cell viability inhibitory concentrations of methanol and aqueous crude extracts of Amaranthus spp. The extracts were screened using small-cell lung cancer (H69V), hepatocellular carcinoma (HepG2/C3A) and non-cancerous kidney cells (Vero) cell lines. Viability was assessed following exposure to a series of concentrations of each extract and A. hypochondriacus showed cytotoxicity of 70.55 µg/mL against H69V with a Si index of 1.8. The fractionated aqueous extract of 40 °C-treated A. hypochondriacus under well-watered conditions had a higher viability inhibition on H69V and Vero cell lines compared to the A. caudatus, A. cruentus and A. spinosus crude extracts. In conclusion, A. hypochondriacus could serve as a potential source of anticancer phytoconstituents for drug development.

2-Aroyl quinazolinone: Synthesis and in vitro anti-parasitic activity.

Trypanosomes and Leishmania are parasitic protozoans that affect millions of people globally. Herein we report the synthesis of 2-aroyl quinazolinones and their antiprotozoal efficacy against Trypanosoma brucei, Trypanosoma brucei rhodesiense, Trypanosoma cruzi, and Leishmania infantum. These compounds were counter-screened against a human cell line for cytotoxicity. Thirteen of the twenty target compounds in this study inhibited the growth of these parasites, with compounds KJ1, and KJ10 exhibiting IC50 values of 4.7 µM (T. b. brucei) and 1.1 µM (T. b. rhodesiense), respectively.

Coproduction and ecological significance of naphthoquinones in carnivorous sundews (Drosera).

While the 1,4-naphthoquinone derivatives 7-methyljuglone (1) and plumbagin (2) possess a diverse and well documented array of biological activities, relatively little remains known about the functional significance of these compounds in planta and, in particular, their possible relation to carnivorous syndromes. In addition, the chemotaxonomic distribution of naphthoquinones (NQs) amongst species of Drosera L. is of phytopharmaceutical interest. Following the quantitative assessment of interspecific variation of 1 and 2 in 13 species and cultivars of Drosera, our findings demonstrate that these NQs are ubiquitously coproduced in, generally, species-specific ratios, and that 1 appears negatively associated with the occurrence of pigmentation in sundews. The prospective antifeedant function of 1 was evaluated in relation to allocation in various organs and ontogenetic phases of D. capensis L., revealing that significantly higher levels were accumulated in young and reproductive organs, most likely for defensive purposes. Investigation into the relationship between the biosynthesis of NQs and carnivory showed that production of 1 is optimally induced and localized in leaves in response to capture of insect prey. As a whole, these findings reveal the clear importance of this secondary metabolite in ecological interactions as well as holding implication for future bioactivity studies on the genus.

Validating a sensitive LCMS method for the quantitation of artemisinin in Artemisia spp. including material used in retracted clinical trials.

Two recent clinical trials reported that Artemisia afra contained significant amounts of the bioactive compound artemisinin. We suspected sample contamination and therefore obtained the A. afra material for testing. A sensitive liquid chromatography mass spectrometry method was developed and validated for the accurate quantitation of artemisinin in Artemisia annua and A. afra plant material. This validated analytical method, with a limit of detection of 0.22 ng/mL (0.22 pg on column), which is an order of magnitude more sensitive than recently published methods, was applied to quantify artemisinin in a collection of Artemisia samples including the A. afra material that was used in the clinical trials.All 16A. annua samples (oldest sample 21 years old) contained the expected levels of artemisinin (0.12-0.63%) whilst none of the A. afra samples in our collection contained any trace of artemisinin (> 0.00001%). However, the A. afra samples used in the clinical trials did contain detectable amounts of artemisinin (0.0013% and 0.0011% vs the claimed amount of 0.0045%).The authors of the clinical trials suspected that cross contamination during sample handling and preparation was likely, reconfirming the importance of having analytical quality control methods in place before clinical trials are conducted. Quality control and ensuring safety of trial participants is of utmost importance.