Hematoxylin, an Alternative Substrate of Tyrosinase.

Mushroom tyrosinase from Agaricus bisporus (abTYR) is often used during the development of tyrosinase inhibitors for medicinal and cosmetic purposes. In the search for novel tyrosinase inhibitors, this study identified hematoxylin as an alternative substrate for abTYR. The interaction of hematoxylin with abTYR was investigated through spectrophotometric and chromatographic analyses. The results showed that hematoxylin acted as an abTYR substrate and exhibited Michaelis-Menten kinetic behaviour at concentrations below 1.25 mM. The substrate properties of hematoxylin were similar to the natural tyrosinase substrate, L-3,4-dihydroxyphenylalanine (L-DOPA), with regards to Km, while Vmax was eightfold lower. The main oxidation product formed during the reaction of abTYR with hematoxylin was identified as hematein. This is the first report of the interaction of hematoxylin with abTYR.

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.

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.

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.

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.

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.

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.

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.

High-throughput screening identifies histone deacetylase inhibitors that modulate GTF2I expression in 7q11.23 microduplication autism spectrum disorder patient-derived cortical neurons.

BACKGROUND: Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental condition affecting almost 1% of children, and represents a major unmet medical need with no effective drug treatment available. Duplication at 7q11.23 (7Dup), encompassing 26-28 genes, is one of the best characterized ASD-causing copy number variations and offers unique translational opportunities, because the hemideletion of the same interval causes Williams-Beuren syndrome (WBS), a condition defined by hypersociability and language strengths, thereby providing a unique reference to validate treatments for the ASD symptoms. In the above-indicated interval at 7q11.23, defined as WBS critical region, several genes, such as GTF2I, BAZ1B, CLIP2 and EIF4H, emerged as critical for their role in the pathogenesis of WBS and 7Dup both from mouse models and human studies. METHODS: We performed a high-throughput screening of 1478 compounds, including central nervous system agents, epigenetic modulators and experimental substances, on patient-derived cortical glutamatergic neurons differentiated from our cohort of induced pluripotent stem cell lines (iPSCs), monitoring the transcriptional modulation of WBS interval genes, with a special focus on GTF2I, in light of its overriding pathogenic role. The hits identified were validated by measuring gene expression by qRT-PCR and the results were confirmed by western blotting. RESULTS: We identified and selected three histone deacetylase inhibitors (HDACi) that decreased the abnormal expression level of GTF2I in 7Dup cortical glutamatergic neurons differentiated from four genetically different iPSC lines. We confirmed this effect also at the protein level. LIMITATIONS: In this study, we did not address the molecular mechanisms whereby HDAC inhibitors act on GTF2I. The lead compounds identified will now need to be advanced to further testing in additional models, including patient-derived brain organoids and mouse models recapitulating the gene imbalances of the 7q11.23 microduplication, in order to validate their efficacy in rescuing phenotypes across multiple functional layers within a translational pipeline towards clinical use. CONCLUSIONS: These results represent a unique opportunity for the development of a specific class of compounds for treating 7Dup and other forms of intellectual disability and autism.

6-Nitro-1-benzylquinolones exhibiting specific antitubercular activity.

In this study, we synthesized novel nitro quinolone-based compounds and tested them in vitro against a panel of Gram-positive and Gram-negative pathogens including Mycobacterium tuberculosis (MTB), Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumonia, Staphylococcus aureus, and Escherichia coli for antibacterial activities and also against HeLa cells for overt cytotoxicity. Compound 8e was identified as a non-toxic, potent hit with selective activity (MIC90  Ë‚ 0.24 µm) against MTB. 8e, however, showed no activity against DprE1 mutant, suggesting DprE1 as the likely target for this compound class.

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.

Artemisia afra, a controversial herbal remedy or a treasure trove of new drugs?

ETHNOPARMACOLOGICAL RELEVANCE: Artemisia afra is one of the most widely used herbal remedies in South Africa. This highly aromatic shrub is used to treat various disorders including coughs, colds, influenza, and malaria. Due to the long tradition of use and popularity of A. afra, it has been successfully commercialised and can currently be bought from various internet stores and pharmacies. The most notable indication is for the prophylaxis and treatment of Plasmodium falciparum infections. In 2013, the Medicine Control Council (MCC) of South Africa banned the sale of A. afra for the treatment of malaria because it lacks scientific evidence of efficacy. This resulted in a lawsuit being filed in 2017 against the MCC by an herbal company which claimed that artemisinin was responsible for A. afra's antiplasmodial activity. At the time, no scientific literature reported that A. afra contained artemisinin. MATERIALS AND METHODS: This review aims to collate all available scientific literature regarding the phytochemistry and biological activity, focusing on antimalarial activity, of A. afra published from 2009 to 2019 and follows on our earlier review, which covered all literature until 2009. All scientific literature in English published between 2009 and June 2019 were retrieved from scientific databases (Scifinder scholar, Web of Science, Scopus, PubMed, Google scholar) and a number of books regarding medicinal plants in South Africa were also consulted. RESULTS: In the last decade very few compounds have been identified in A. afra, none of which were novel compounds. Based on all the tests that have been conducted using extracts and compounds of A. afra in a disparate variety of in vitro and in vivo bioassays, the results indicate only weak biological activity. The activity of extracts, and in some cases pure compounds, exhibited IC50 or MIC values of 1000-10 000 fold less active than the positive controls. In contrast, and quite surprisingly, two randomised controlled trials were recently conducted (Schistosoma mansoni and Plasmodium falciparum infected patients) and although criticised based on design, execution, statistical analysis and ethical concerns, showed remarkably positive results. CONCLUSIONS: Pre-clinical in vitro and in vivo animal experiments failed to yield any promising drug leads. However, if the recent randomised controlled trials can be independently replicated in well-designed and executed clinical trials it might indicate that A. afra contain powerful 'prodrugs'. Future research on A. afra should therefore focus on reproducing the randomised controlled trials and on artificially metabolising A. afra extracts/compounds in order to identify the presence of any 'prodrugs'.

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.

Novel BRPF1 mutation in a boy with intellectual disability, coloboma, facial nerve palsy and hypoplasia of the corpus callosum.

Mutations in the chromatin regulator gene BRPF1 were recently associated with the Intellectual Developmental Disorder With Dysmorphic Facies And Ptosis (IDDDFP). Up till now, clinical data of 22 patients are reported. Besides intellectual disability (ID), ptosis and blepharophimosis are frequent findings, with refraction problems, amblyopia and strabism as other reported ophthalmological features. Animal studies indicate BRPF1 as an important mediator in brain development. However, only 5 of 22 previously reported patients show structural brain abnormalities. We report on an additional patient harboring a novel de novo nonsense mutation p.(Glu219*) in BRPF1. He presented with ID, bilateral iris colobomas, facial nerve palsy and severe hypoplasia of the corpus callosum. Our findings support previous findings of brain abnormalities in BRPF1-mutations and indicates coloboma and facial nerve palsy as possible additional features of IDDDFP syndrome.

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.

A mouse model for intellectual disability caused by mutations in the X-linked 2'­O­methyltransferase Ftsj1 gene.

Mutations in the X chromosomal tRNA 2'­O­methyltransferase FTSJ1 cause intellectual disability (ID). Although the gene is ubiquitously expressed affected individuals present no consistent clinical features beyond ID. In order to study the pathological mechanism involved in the aetiology of FTSJ1 deficiency-related cognitive impairment, we generated and characterized an Ftsj1 deficient mouse line based on the gene trapped stem cell line RRD143. Apart from an impaired learning capacity these mice presented with several statistically significantly altered features related to behaviour, pain sensing, bone and energy metabolism, the immune and the hormone system as well as gene expression. These findings show that Ftsj1 deficiency in mammals is not phenotypically restricted to the brain but affects various organ systems. Re-examination of ID patients with FTSJ1 mutations from two previously reported families showed that several features observed in the mouse model were recapitulated in some of the patients. Though the clinical spectrum related to Ftsj1 deficiency in mouse and man is variable, we suggest that an increased pain threshold may be more common in patients with FTSJ1 deficiency. Our findings demonstrate novel roles for Ftsj1 in maintaining proper cellular and tissue functions in a mammalian organism.