Curcumin: A naturally occurring autophagy modulator

Autophagy is a self-degradative process that plays a pivotal role in several medical conditions associated with infection, cancer, neurodegeneration, aging, and metabolic disorders. Its interplay with cancer development and treatment resistance is complicated and paramount for drug design since an autophagic response can lead to tumor suppression by enhancing cellular integrity and tumorigenesis by improving tumor cell survival. In addition, autophagy denotes the cellular ability of adapting to stress though it may end up in apoptosis activation when cells are exposed to a very powerful stress. Induction of autophagy is a therapeutic option in cancer and many anticancer drugs have been developed to this aim. Curcumin as a hydrophobic polyphenol compound extracted from the known spice turmeric has different pharmacological effects in both in vitro and in vivo models. Many reports exist reporting that curcumin is capable of triggering autophagy in several cancer cells. In this review, we will focus on how curcumin can target autophagy in different cellular settings that may extend our understanding of new pharmacological agents to overcome relevant diseases.     

Effects of polyploidization on petal characteristics and optical properties of Impatiens walleriana (Hook.)

Plant Cell, Tissue and Organ Culture (PCTOC) - Impatiens walleriana (Hook.) (2n?=?2x?=?16) is a popular flowering species in the Balsaminaceae family. In order to...     

Optimizing the attractiveness of pheromone baits used for trapping the four‐eyed spruce bark beetle Polygraphus poligraphus

Bark beetles have caused extensive damage to forests in central Sweden during the past decade, and the four‐eyed spruce bark beetle, Polygraphus poligraphus, seems to be involved. However, its role in these bark beetle outbreaks is still not clear. The purpose of this study was to develop an efficient pheromone bait for P. poligraphus, which would make it possible to study the species more carefully and thereby contribute to protect exposed forests in an environmentally friendly way. Three field studies were conducted in 2015, 2016 and 2018 in Medelpad, county of Västernorrland, Sweden. The pheromone of P. poligraphus, (?)‐terpinen‐4‐ol, was tested at different release rates and in different enantiomeric purities, to find the most attractive formulation for the beetles. It was also tested in combination with racemic frontalin, a compound which has previously been shown to produce a synergistic effect together with (?)‐terpinen‐4‐ol of low enantiomeric purity; 52% ee. Other compounds, chosen based on responses from electroantennographic studies, were also tested in an attempt to find additional attractants and repellents for P. poligraphus. The most attractive treatment tested was enantiomerically pure (?)‐terpinen‐4‐ol (99% ee). When the enantiomeric purity was lower (50% ee), the trap catches was lowered to levels comparable to the catches for unbaited control traps. A strong synergistic effect with frontalin was observed for (?)‐terpinen‐4‐ol of low enantiomeric purity (50% ee) but not for the enantiomerically pure compound (99% ee). The release rate of (?)‐terpinen‐4‐ol (99% ee) was shown to be an important factor. For the combination of frontalin and (?)‐terpinen‐4‐ol (50% ee), the attraction seemed strongest when (?)‐terpinen‐4‐ol was released at a higher rate than frontalin. An interesting and novel result was that a repellent compound, α‐terpineol, was identified in our studies. Our results from field studies and electroantennography recordings also indicate that (+)‐terpinen‐4‐ol is a repellent for P. poligraphus.     

Possible correlation between high circulatory levels of trimethylamine-N-oxide and 2177G>C polymorphisms of hepatic flavin containing monooxygenase 3 in Kurdish Population with non-alcoholic fatty liver disease

Molecular Biology Reports - Non-alcoholic fatty liver disease (NAFLD) is a multifactorial disorder with complicated pathophysiology. Trimethylamine-N-oxide (TMAO) has been thought to be correlated...     

Energetic adaptations: Metabolic control of endocytic membrane traffic

Endocytic membrane traffic controls the access of myriad cell surface proteins to the extracellular milieu, and thus gates nutrient uptake, ion homeostasis, signaling, adhesion and migration. Coordination of the regulation of endocytic membrane traffic with a cell's metabolic needs represents an important facet of maintenance of homeostasis under variable conditions of nutrient availability and metabolic demand. Many studies have revealed intimate regulation of endocytic membrane traffic by metabolic cues, from the specific control of certain receptors or transporters, to broader adaptation or remodeling of the endocytic membrane network. We examine how metabolic sensors such as AMP‐activated protein kinase, mechanistic target of rapamycin complex 1 and hypoxia inducible factor 1 determine sufficiency of various metabolites, and in turn modulate cellular functions that includes control of endocytic membrane traffic. We also examine how certain metabolites can directly control endocytic traffic proteins, such as the regulation of specific protein glycosylation by limiting levels of uridine diphosphate N‐acetylglucosamine (UDP‐GlcNAc) produced by the hexosamine biosynthetic pathway. From these ideas emerge a growing appreciation that endocytic membrane traffic is orchestrated by many intrinsic signals derived from cell metabolism, allowing alignment of the functions of cell surface proteins with cellular metabolic requirements. Endocytic membrane traffic determines how cells interact with their environment, thus defining many aspects of nutrient uptake and energy consumption. We examine how intrinsic signals that reflect metabolic status of a cell regulate endocytic traffic of specific proteins, and, in some cases, exert broad control of endocytic membrane traffic phenomena. Hence, endocytic traffic is versatile and adaptable and can be modulated to meet the changing metabolic requirements of a cell.     

Comparative effects of rifabutin and rifampicin on cytochromes P450 and UDP-glucuronosyl-transferases expression in fresh and cryopreserved human hepatocytes.

The aim of this study was to evaluate rifabutin (RBT) and rifampicin (RIF) capabilities in inducing various xenobiotic metabolizing enzymes such as cytochromes P450 (CYPs) and UDP-glucuronosyl-transferases (UGTs) in cultured fresh and cryopreserved human hepatocytes. Enzyme induction was assessed through the use of several diagnostic markers, i.e. testosterone, midazolam (MDZ), diazepam (DZP) and 7-ethoxyresorufin for CYP-dependent enzyme reactions; and AZT for UGT-dependent enzyme reactions. RBT concentrations (0.118, 0.708 microM) were selected according to previously published pharmacokinetic data in patients. The known CYP3A4 inducer in humans, RIF, was used as a positive control. At the concentrations used, no sign of cytotoxicity was evidenced. Both compounds were able to dose-dependently induce the overall metabolism of testosterone (approximately 2-fold for RBT, 4-fold for RIF) and the formation of the 6beta-hydroxylated-derivative (up to approximately 4-fold over control for RBT and approximately 10-fold for RIF), which is CYP3A4 dependent. The other hydroxylated metabolites (16alpha-OH and 2alpha-OH) were also enhanced. The metabolism of MDZ, which is specifically metabolized by CYP3A4 in humans, was also investigated following drug's exposure to hepatocytes. DZP one, which is governed by various CYPs, including CYP3A, was also investigated. RBT was shown to increase the biotransformation of both benzodiazepines (approximately 1.9-fold over control). Moreover, the effects of both drugs on ethoxyresorufin O-deethylase activity (EROD), which is representative of CYPIA1/2 isoforms, were tested. Results showed only a moderate induction of this marker (approximately 2-fold over control) when compared to the high effect observed after hepatocyte exposure to 3-methylcholantene (approximately 14-fold over control). Finally, the action of RBT and RIF on UGTs expression was investigated by using AZT as diagnostic substrate: glucuronides formation was not significantly affected by the two rifamycin derivatives. On the whole, exposure of fresh or cryopreserved human hepatocytes to RBT dose-dependently affected the levels of drug metabolizing enzymes in a dose-dependent manner. However, as already demonstrated by in vivo pharmacokinetic studies, its inducing properties towards CYPs, CYP3A in particular, are less pronounced than RIF.     

A multi-laboratory evaluation of cryopreserved monkey hepatocyte functions for use in pharmaco-toxicology.

Ethical, economic and technical reasons hinder regular supply of freshly isolated hepatocytes from higher mammals such as monkey for preclinical evaluation of drugs. Hence, we aimed at developing optimal and reproducible protocols to cryopreserve and thaw parenchymal liver cells from this major toxicological species. Before the routine use of these protocols, we validated them through a multi-laboratory study. Dissociation of the whole animal liver resulted in obtaining 1-5 billion parenchymal cells with a viability of about 86%. An appropriate fraction (around 20%) of the freshly isolated cells was immediately set in primary culture and various hepato-specific tests were performed to examine their metabolic, biochemical and toxicological functions as well as their ultrastructural characteristics. The major part of the hepatocytes was frozen and their functionality checked using the same parameters after thawing. The characterization of fresh and thawed monkey hepatocytes demonstrated the maintenance of various hepato-specific functions. Indeed, cryopreserved hepatocytes were able to survive and to function in culture as well as their fresh counterparts. The ability for synthesis (proteins, ATP, GSH) and conjugation and secretion of biliary acids was preserved after deep freeze storage. A better stability of drug metabolizing activities than in rodent hepatocytes was observed in monkey. After thawing, Phase I and Phase II activities (cytochrome P450, ethoxycoumarin-O-deethylase, aldrin epoxidase, epoxide hydrolase, glutathione transferase, glutathione reductase and glutathione peroxidase) were well preserved. The metabolic patterns of several drugs were qualitatively and quantitatively similar before and after cryopreservation. Lastly, cytotoxicity tests suggested that the freezing/thawing steps did not change cell sensitivity to toxic compounds.     

Mosaicism in CRISPR/Cas9-mediated genome editing

The CRISPR/Cas9 system is a rapid, simple, and often extremely efficient gene editing method. This method has been used in a variety of organisms and cell types over the past several years. However, using this technology for generating gene-edited animals involves a number of obstacles. One such obstacle is mosaicism, which is common in founder animals. This is especially the case when the CRISPR/Cas9 system is used in embryos. Here we review the pros and cons of mosaic mutations of gene-edited animals caused by using the CRISPR/Cas9 system in embryos. Furthermore, we will discuss the mechanisms underlying mosaic mutations resulting from the CRISPR/Cas9 system, as well as the possible strategies for reducing mosaicism. By developing ways to overcome mosaic mutations when using CRISPR/Cas9, genotyping for germline gene disruptions should become more reliable. This achievement will pave the way for using the CRISPR technology in the research and clinical applications where mosaicism is an issue.     

Slot blot method for the quantification of DNA sequences and mapping of chromosome rearrangements: Application to chromosome 21

As an alternative to the methods of gene dosage based on either RFLP studies or Southern blots using specific and reference probes, we designed a "slot blot" method for the evaluation of the copy number of unique chromosome 21 sequences. Varying amounts of denatured DNA from a normal control, a trisomy 21 patient, and the subject to be analyzed were loaded on the same membrane. Successive hybridizations with reference probes and chromosome 21 probes were then carried out. Intensities of the signals on autoradiograms were quantified by densitometric scanning. Graphic and statistical analysis of the linear regressions between reference and chromosome 21 probe signals were performed, and the conclusion that the DNA from the studied subject had two or three copies for a given chromosome 21 sequence was assessed by statistical comparison of the slopes. As a test for the validation of this method, 10 coded blood DNAs from five normal controls and from five trisomy 21 patients were analyzed, by using two reference (COL1A1 and COL1A2) and two chromosome 21 (D21S11 and D21S17) probes. Among the 10 DNAs analyzed, it was possible to diagnose, with 100% accuracy, normal controls and trisomic 21 individuals. Application of this methodology to the mapping of partial chromosome 21 rearrangements is presented.     

Gene-dosage mapping of 30 DNA markers on chromosome 21.

Using a slot-blot method for the dosage of single-copy sequences, the copy numbers of 30 chromosome 21 markers were assessed in the blood DNA of 11 patients with partial trisomy or monosomy 21 and in the DNA of a patient-derived human-hamster hybrid cell line carrying a microduplication of chromosome 21. The physical order of these markers on chromosome 21 was thereby determined.