Anti-acetylcholinesterase activity and antioxidant properties of extracts and fractions of Carpolobia lutea.

CONTEXT: There is an unmet need to discover new treatments for Alzheimer's disease. This study determined the anti-acetylcholinesterase (AChE) activity, DPPH free radical scavenging and antioxidant properties of Carpolobia lutea G. Don (Polygalaceae). OBJECTIVE: The objective of this study is to quantify C. lutea anti-AChE, DPPH free radical scavenging, and antioxidant activities and cell cytotoxicity. MATERIALS AND METHODS: Plant stem, leaves and roots were subjected to sequential solvent extractions, and screened for anti-AChE activity across a concentration range of 0.02-200 µg/mL. Plant DPPH radical scavenging activity, reducing power, and total phenolic and flavonoid contents were determined, and cytotoxicity evaluated using human hepatocytes. RESULTS: Carpolobia lutea exhibited concentration-dependent anti-AChE activity. The most potent inhibitory activity for the stem was the crude ethanol extract and hexane stem fraction oil (IC50 = 140 µg/mL); for the leaves, the chloroform leaf fraction (IC50 = 60 µg/mL); and for roots, the methanol, ethyl acetate and aqueous root fractions (IC50 = 0.3-3 µg/mL). Dose-dependent free radical scavenging activity and reducing power were observed with increasing stem, leaf or root concentration. Total phenolic contents were the highest in the stem: ∼632 mg gallic acid equivalents/g for a hexane stem fraction oil. Total flavonoid content was the highest in the leaves: ∼297 mg quercetin equivalents/g for a chloroform leaf fraction. At 1 µg/mL, only the crude ethanol extract oil was significantly cytotoxic to hepatocytes. DISCUSSION AND CONCLUSIONS: Carpolobia lutea possesses anti-AChE activity and beneficial antioxidant capacity indicative of its potential development as a treatment of Alzheimer's and other diseases characterized by a cholinergic deficit.

Comparative effects of viral-transport-medium heat inactivation upon downstream SARS-CoV-2 detection in patient samples.

Introduction. The COVID-19 pandemic, which began in 2020 is testing economic resilience and surge capacity of healthcare providers worldwide. At the time of writing, positive detection of the SARS-CoV-2 virus remains the only method for diagnosing COVID-19 infection. Rapid upscaling of national SARS-CoV-2 genome testing presented challenges: (1) Unpredictable supply chains of reagents and kits for virus inactivation, RNA extraction and PCR-detection of viral genomes. (2) Rapid time to result of <24 h is required in order to facilitate timely infection control measures.Hypothesis. Extraction-free sample processing would impact commercially available SARS-CoV-2 genome detection methods.Aim. We evaluated whether alternative commercially available kits provided sensitivity and accuracy of SARS-CoV-2 genome detection comparable to those used by regional National Healthcare Services (NHS).Methodology. We tested several detection methods and tested whether detection was altered by heat inactivation, an approach for rapid one-step viral inactivation and RNA extraction without chemicals or kits.Results. Using purified RNA, we found the CerTest VIASURE kit to be comparable to the Altona RealStar system currently in use, and further showed that both diagnostic kits performed similarly in the BioRad CFX96 and Roche LightCycler 480 II machines. Additionally, both kits were comparable to a third alternative using a combination of Quantabio qScript one-step Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) mix and Centre for Disease Control and Prevention (CDC)-accredited N1 and N2 primer/probes when looking specifically at borderline samples. Importantly, when using the kits in an extraction-free protocol, following heat inactivation, we saw differing results, with the combined Quantabio-CDC assay showing superior accuracy and sensitivity. In particular, detection using the CDC N2 probe following the extraction-free protocol was highly correlated to results generated with the same probe following RNA extraction and reported clinically (n=127; R2=0.9259).Conclusion. Our results demonstrate that sample treatment can greatly affect the downstream performance of SARS-CoV-2 diagnostic kits, with varying impact depending on the kit. We also showed that one-step heat-inactivation methods could reduce time from swab receipt to outcome of test result. Combined, these findings present alternatives to the protocols in use and can serve to alleviate any arising supply-chain issues at different points in the workflow, whilst accelerating testing, and reducing cost and environmental impact.

Excess androgen production in subcutaneous adipose tissue of women with polycystic ovarian syndrome is not related to insulin or LH.

The purpose of this study was to investigate androgen production and the role of insulin and LH in its regulation in subcutaneous adipose tissue (SAT) of women with polycystic ovarian syndrome (PCOS). Protein and mRNA expression of androgen synthesis enzymes (Cytochrome P450 17A1 [CYP17A1] and Aldo-keto reductase 1C3 [AKR1C3]) were measured in SAT biopsies from women with PCOS, diagnosed according to the Rotterdam criteria (n=15) and healthy controls (n=15). Cultured mature adipocytes (differentiated from SAT biopsies) were treated with insulin ± phosphoinositol-3-kinase inhibitor (LY294002) or LH ± insulin. CYP17A1 and AKR1C3 mRNA expression and testosterone concentrations were measured in treated and untreated adipocyte cultures. AKR1C3 mRNA was significantly (P<0.001) greater in PCOS versus non-PCOS SAT, but CYP17A1 was not significantly different between the two groups. AKR1C3 and CYP17A1 protein expression was not significantly different in PCOS versus non-PCOS SAT. In untreated adipocyte cultures, CYP17A1, AKR1C3 and testosterone levels were significantly higher in the PCOS versus the non-PCOS groups. Addition of insulin increased AKR1C3 mRNA and testosterone levels, but not CYP17A1 mRNA in non-PCOS with no effect on PCOS adipocytes. The stimulatory effects of insulin were not inhibited by LY294002. Addition of LH increased CYP17A1, AKR1C3 and testosterone in non-PCOS adipocytes with no effect in PCOS adipocytes. In conclusion, SAT of women with PCOS produces excess androgen, which may contribute to PCOS-related hyperandrogenaemia. This SAT androgen excess is independent of obesity and is not directly stimulated by insulin or LH.

The Loss of α- and ß-Tubulin Proteins Are a Pathological Hallmark of Chronic Alcohol Consumption and Natural Brain Ageing.

Repetitive excessive alcohol intoxication leads to neuronal damage and brain shrinkage. We examined cytoskeletal protein expression in human post-mortem tissue from Brodmann's area 9 of the prefrontal cortex (PFC). Brain samples from 44 individuals were divided into equal groups of 11 control, 11 alcoholic, 11 non-alcoholic suicides, and 11 suicide alcoholics matched for age, sex, and post-mortem delay. Tissue from alcoholic cohorts displayed significantly reduced expression of α- and ß-tubulins, and increased levels of acetylated α-tubulin. Protein levels of histone deacetylase-6 (HDAC6), and the microtubule-associated proteins MAP-2 and MAP-tau were reduced in alcoholic cohorts, although for MAPs this was not significant. Tubulin gene expressions increased in alcoholic cohorts but not significantly. Brains from rats administered alcohol for 4 weeks also displayed significantly reduced tubulin protein levels and increased α-tubulin acetylation. PFC tissue from control subjects had reduced tubulin protein expression that was most notable from the sixth to the eighth decade of life. Collectively, loss of neuronal tubulin proteins are a hallmark of both chronic alcohol consumption and natural brain ageing. The reduction of cytosolic tubulin proteins could contribute to the brain volumetric losses reported for alcoholic patients and the elderly.

Inflammation and MiR-21 pathways functionally interact to downregulate PDCD4 in colorectal cancer.

Inflammation plays a direct role in colorectal cancer (CRC) progression; however the molecular mechanisms responsible for this effect are unclear. The inflammation induced cyclooxygenase 2 (COX-2) enzyme required for the production of Prostaglandin E2 (PGE2), can promote colorectal cancer by decreasing expression of the tumour suppressor gene Programmed Cell Death 4 (PDCD4). As PDCD4 is also a direct target of the oncogene microRNA-21 (miR-21) we investigated the relationship between the COX-2 and miR-21 pathways in colorectal cancer progression. Gene expression profile in tumour and paired normal mucosa from 45 CRC patients demonstrated that up-regulation of COX-2 and miR-21 in tumour tissue correlates with worse Dukes' stage. In vitro studies in colonic adenocarcinoma cells revealed that treatment with the selective COX-2 inhibitor NS398 significantly decreased miR-21 levels (p = 0.0067) and increased PDCD4 protein levels (p<0.001), whilst treatment with PGE2 up-regulated miR-21 expression (p = 0.019) and down-regulated PDCD4 protein (p<0.05). These findings indicate that miR-21 is a component of the COX-2 inflammation pathway and that this pathway promotes worsening of disease stage in colorectal cancer by inducing accumulation of PGE2 and increasing expression of miR-21 with consequent downregulation of the tumour suppressor gene PDCD4.