Lipoprotein (a) in familial hypercholesterolaemia.

PURPOSE OF REVIEW: The role of lipoprotein (a) in atherogenesis has been the subject of argument for many years. Evidence that it is raised in familial hypercholesterolaemia has been disputed not least because a mechanism related to low density lipoprotein (LDL) receptor mediated catabolism has been lacking. Whether lipoprotein (a) increases the already raised atherosclerotic cardiovascular disease (ASCVD) risk in familial hypercholesterolaemia is also more dubious than is often stated. We review the evidence in an attempt to provide greater clarity. RECENT FINDINGS: Lipoprotein (a) levels are raised as a consequence of inheriting familial hypercholesterolaemia. The mechanism for this is likely to involve increased hepatic production, probably mediated by PCSK9 augmented by apolipoprotein E. The extent to which raised lipoprotein (a) contributes to the increased ASCVD risk in familial hypercholesterolaemia remains controversial.Unlike, for example, statins which are effective across the whole spectrum of LDL concentrations, drugs in development to specifically lower lipoprotein (a) are likely to be most effective in people with the highest levels of lipoprotein (a). People with familial hypercholesterolaemia may therefore be in the vanguard of those in whom theses agents should be exhibited. SUMMARY: Inheritance of familial hypercholesterolaemia undoubtedly increases the likelihood that lipoprotein (a) will be raised. However, in familial hypercholesterolaemia when ASCVD incidence is already greatly increased due to high LDL cholesterol, whether lipoprotein (a) contributes further to this risk cogently needs to be tested with drugs designed to specifically lower lipoprotein (a).

Recent developments and applications of hyperspectral imaging for rapid detection of mycotoxins and mycotoxigenic fungi in food products.

Mycotoxins are the foremost naturally occurring contaminants of food products such as corn, peanuts, tree nuts, and wheat. As the secondary metabolites, mycotoxins are mainly synthesized by many species of the genera Aspergillus, Fusarium and Penicillium, and are considered highly toxic and carcinogenic to humans and animals. Most mycotoxins are detected and quantified by analytical chemistry-based methods. While mycotoxigenic fungi are usually identified and quantified by biological methods. However, these methods are time-consuming, laborious, costly, and inconsistent because of the variability of the grain-sampling process. It is desirable to develop rapid, non-destructive and efficient methods that objectively measure and evaluate mycotoxins and mycotoxigenic fungi in food. In recent years, some spectroscopy-based technologies such as hyperspectral imaging (HSI), Raman spectroscopy, and Fourier transform infrared spectroscopy have been extensively investigated for their potential use as tools for the detection, classification, and sorting of mycotoxins and toxigenic fungal contaminants in food. HSI integrates both spatial and spectral information for every pixel in an image, making it suitable for rapid detection of large quantities of samples and more heterogeneous samples and for in-line sorting in the food industry. In order to track the latest research developments in HSI, this paper gives a brief overview of the theories and fundamentals behind the technology and discusses its applications in the field of rapid detection and sorting of mycotoxins and toxigenic fungi in food products. Additionally, advantages and disadvantages of HSI are compared, and its potential use in commercial applications is reported.

The bZIP transcription factor Afap1 mediates the oxidative stress response and aflatoxin biosynthesis in Aspergillus flavus.

Aflatoxin is a carcinogenic secondary metabolite produced mainly by Aspergillus flavus and Aspergillus parasiticus, which can seriously endanger the health of humans and animals. Oxidative stress is a common defense response, and it is known that reactive oxygen species (ROS) can induce the synthesis of a series of secondary metabolites, including aflatoxin. By using mutants lacking the afap 1 gene, the role of afap1 gene in oxidative stress and aflatoxin synthesis was assessed. The growth of the mutant strains was significantly inhibited by the increase in the concentration of H2O2, inhibition was complete at 40mmol/l. However, in the quantitative analysis by HPLC, the concentration of AFB1 increased with the increased H2O2 until 10mmol/l. Following an analysis based on the information provided by the NCBI BLAST analysis, it was assumed that Afap1, a basic leucine zipper (bZIP) transcription factor, was associated with the oxidative stress in this fungus. Treatment with 5mmol/l H2O2 completely inhibited the growth of the mutant strains in afap 1 but did not affect the growth of the CA14PTs strain (non-mutant strain). In addition, the concentration of AFB1 in the mutant strains was approximately » of that observed in the CA14PTs strain. These results suggested that Afap1 plays a key role in the regulation of oxidative stress and aflatoxin production in A. flavus.

Monitoring Metabolite Production of Aflatoxin Biosynthesis by Orbitrap Fusion Mass Spectrometry and a D-Optimal Mixture Design Method.

Aflatoxins, highly toxic and carcinogenic to humans, are synthesized via multiple intermediates by a complex pathway in several Aspergilli, including Aspergillus flavus. Few analytical methods are available for monitoring the changes in metabolite profiles of the aflatoxin biosynthesis pathway under different growth and environmental conditions. In the present study, we developed by a D-optimal mixture design a solvent system, methanol/dichloromethane/ethyl acetate/formic acid (0.36/0.31/0.32/0.01), that was suitable for extracting the pathway metabolites. The matrix effect from dilution of cell extracts was negligible. To facilitate the identification of these metabolites, we constructed a fragmentation ion library. We further employed liquid chromatography coupled with high-resolution mass spectroscopy (UHPLC-HRMS) for simultaneous quantification of the metabolites. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.002-0.016 and 0.008-0.05 µg/kg, respectively. The spiked recovery rates ranged from 81.3 to 100.3% with intraday and interday precision less than 7.6%. Using the method developed to investigate the time-course aflatoxin biosynthesis, we found that precursors, including several possible toxins (with a carcinogenic group similar to aflatoxin B1), occurred together with aflatoxin, and that production increased rapidly at the early growth stage, peaked on day four, and then decreased substantially. The maximum production of aflatoxin B1 and aflatoxin B2 occurred 1 day later. Moreover, the dominant branch pathway was the one for aflatoxin B1 formation. We revealed that the antiaflatoxigenicity mechanism of Leclercia adecarboxylata WT16 was associated with a factor upstream of the aflatoxin biosynthesis pathway. The design strategies can be applied to characterize or detect other secondary metabolites to provide a snapshot of the dynamic changes during their biosynthesis.

Peanuts that keep aflatoxin at bay: a threshold that matters.

Aflatoxin contamination in peanuts poses major challenges for vulnerable populations of sub-Saharan Africa and South Asia. Developing peanut varieties to combat preharvest Aspergillus flavus infection and resulting aflatoxin contamination has thus far remained a major challenge, confounded by highly complex peanut-Aspergilli pathosystem. Our study reports achieving a high level of resistance in peanut by overexpressing (OE) antifungal plant defensins MsDef1 and MtDef4.2, and through host-induced gene silencing (HIGS) of aflM and aflP genes from the aflatoxin biosynthetic pathway. While the former improves genetic resistance to A. flavus infection, the latter inhibits aflatoxin production in the event of infection providing durable resistance against different Aspergillus flavus morphotypes and negligible aflatoxin content in several peanut events/lines well. A strong positive correlation was observed between aflatoxin accumulation and decline in transcription of the aflatoxin biosynthetic pathway genes in both OE-Def and HIGS lines. Transcriptomic signatures in the resistant lines revealed key mechanisms such as regulation of aflatoxin synthesis, its packaging and export control, besides the role of reactive oxygen species-scavenging enzymes that render enhanced protection in the OE and HIGS lines. This is the first study to demonstrate highly effective biotechnological strategies for successfully generating peanuts that are near-immune to aflatoxin contamination, offering a panacea for serious food safety, health and trade issues in the semi-arid regions.

Identification of candidate resistance genes of cotton against Aspergillus flavus infection using a comparative transcriptomics approach.

A comparative transcriptome analysis was performed using the genes significantly differentially expressed in cotton, corn and peanut in response to aflatoxin producing fungus Aspergillus flavus with an objective of identifying candidate resistance genes in cotton. Two-way analyses identified 732 unique genes to be differentially regulated by the fungus with only 26 genes common across all three crops that were considered candidate A. flavus resistance genes with an assumption that these genes have specific roles in conferring the resistance trait. Genes of membrane cellular component involved in DNA binding with involvement in defense responses were highly represented among the differentially expressed unique genes. Most (six) of these genes coded for 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily proteins. Genes encoding helix loop helix protein, alcohol dehydrogenase and UDP glycosylation transferase which were upregulated in response to both atoxigenic and toxigenic strains of A. flavus, could be potential resistance candidate genes for downstream functional manipulation to confer resistance.

Crystal structure and biochemical investigations reveal novel mode of substrate selectivity and illuminate substrate inhibition and allostericity in a subfamily of Xaa-Pro dipeptidases.

Xaa-Pro dipeptidase (XPD) catalyzes hydrolysis of iminopeptide bond in dipeptides containing trans-proline as a second residue. XPDs are found in all living organisms and are believed to play an essential role in proline metabolism. Here, we report crystal structures and extensive enzymatic studies of XPD from Xanthomonas campestris (XPDxc), the first such comprehensive study of a bacterial XPD. We also report enzymatic activities of its ortholog from Mycobacterium tuberculosis (XPDmt). These enzymes are strictly dipeptidases with broad substrate specificities. They exhibit substrate inhibition and allostericity, as described earlier for XPD from Lactococcus lactis (XPDll). The structural, mutational and comparative data have revealed a novel mechanism of dipeptide selectivity and substrate binding in these enzymes. Moreover, we have identified conserved sequence motifs that distinguish these enzymes from other prolidases, thus defining a new subfamily. This study provides a suitable structural template for explaining unique properties of this XPDxc subfamily. In addition, we report unique structural features of XPDxc protein like an extended N-terminal tail region and absence of a conserved Tyr residue near the active site.

The TLR4-NOS1-AP1 signaling axis regulates macrophage polarization.

OBJECTIVE: Macrophages polarize to proinflammatory M1 or anti-inflammatory M2 states with distinct physiological functions. This transition within the M1-M2 phenotypes decides the nature, duration and severity of an inflammatory response. Although there is a substantial understanding of the fate of these phenotypes, the underlying molecular mechanism of transition within the M1-M2 phenotypes is not well understood. We have investigated the role of neuronal nitric oxide synthase (NOS1)-mediated regulation of activator protein 1 (AP-1) transcription factor in macrophages as a critical effector of macrophage phenotypic change. MATERIALS AND METHODS: Raw 264.7 and THP1 macrophages were stimulated with LPS (250 ng/ml) to activate the inflammatory signaling pathway. We analyzed the effect of pharmacological NOS1 inhibitor: TRIM (1-(2- Trifluoromethylphenyl) imidazole) on LPS-induced inflammatory response in macrophages. RESULTS: We determined that NOS1-derived nitric oxide (NO) facilitate Fos and Jun interaction which induces IL-12 & IL-23 expression. Pharmacological inhibition of NOS1 inhibits ATF2 and Jun dimer. Switching of Fos and Jun dimer to ATF2 and Jun dimerization controls phenotype transition from IL-12high IL-23high IL-10low to IL-12low IL-23lowIL-10high phenotype, respectively. CONCLUSION: These findings highlight a key role of the TLR4-NOS1-AP1 signaling axis in regulating macrophage polarization.

Amelioration of oxidative and inflammatory changes by Swertia chirayita leaves in experimental arthritis.

The present study was aimed to determine the therapeutic effects of Swertia chirayita leaves against oxidative and inflammatory injuries in Freund's complete adjuvant (FCA) induced arthritic rats. The extract was evaluated for its phytoconstituents and various invitro antioxidant properties followed by its in vivo effects. The hydroethanolic extract of S. chirayita leaves (SCE) was orally administered (200 mg/kg body weight, per day, p.o.) and the effect on the liver lipid peroxidation (LPO), antioxidant status, protein carbonyl formation along with the histopathology of liver were evaluated after induction of adjuvant arthritis. The markers of inflammation and arthritis, such as tumor necrosis factor-α (TNF-α), interleukin 1α (IL-1α), inhibition of paw edema, along with the histological and radiographic changes in the arthritic ankle joint were studied with and without SCE administration. The result showed the presence of major phytoconstituents, such as phenolic, flavonoid and terpenoid content in SCE. HPLC analysis revealed the presence of swertiamarin and amarogentin in high concentration. The extract also showed in vitro antioxidant potential which has positive correlation with the phytoconstituents. The result of in vivo study showed elevated malondialdehyde (MDA) and carbonyl content indicative of LPO and protein oxidation, respectively, with compromised intracellular antioxidant defense system in arthritic rats, which were significantly normalized after SCE treatment. The increase in serum proinflammatory cytokines (TNF- α and IL-1α) and paw edema of arthritic rats was significantly suppressed by SCE. Histology and radiographic analysis of arthritic ankle joints indicated abnormal changes. Marked reduction in inflammation and arthritic changes were observed after treatment with SCE. The present investigation suggests that hydroethanolic extract of S. chirayita leaves exhibit potential immunomodulatory effects, which may possibly be due to boosting the intracellular antioxidant defense.

Nuclear heterogeneity in conidial populations of Aspergillus flavus.

Aspergillus flavus is a major producer of aflatoxin and an opportunistic pathogen for a wide range of hosts. Understanding genotypic and phenotypic variation within strains of A. flavus is important for controlling disease and reducing aflatoxin contamination. A. flavus is multinucleate and predominantly haploid (n) and homokaryotic. Although cryptic heterokaryosis may occur in nature, it is unclear how nuclei in A. flavus influence genetic heterogeneity and if nuclear condition plays a role in fungal ecology. A. flavus mainly reproduces asexually by producing conidia. In order to observe whether conidia are homokaryotic or heterokaryotic, we labeled nuclei of A. flavus using two different nuclear localized fluorescent reporters. The reporter constructs (pYH2A and pCH2B), encode histones HH2A and HH2B fused at the C terminus with either yellow (EYFP) or cyan (ECFP) fluorescent proteins, respectively. The constructs were transformed into the double auxotrophic strain AFC-1 (-pyrG, -argD) to generate a strain containing each reporter construct. By taking advantage of the nutritional requirement for each strain, we were able to generate fusants between FR36 (-argD) expressing yellow fluorescence, and FR46 (-pyr4) expressing cyan fluorescence. Conidia from fusants between FR36 and FR46 showed three types of fluorescence: only EYFP, only ECFP or both EYFP+ECFP. Conidia containing nuclei expressing EYFP+ECFP were separated by Fluorescence-Activated Cell sorting (FACS) and were found to contain both yellow and cyan fluorescent markers in the same nucleus. Further characterization of conidia having only one nucleus but expressing both EYFP+ECFP fluorescence were found to be diploid (2n). Our findings suggest that A. flavus maintains nuclear heterogeneity in conidial populations.

An Aspergillus flavus secondary metabolic gene cluster containing a hybrid PKS-NRPS is necessary for synthesis of the 2-pyridones, leporins.

The genome of the filamentous fungus, Aspergillus flavus, has been shown to harbor as many as 56 putative secondary metabolic gene clusters including the one responsible for production of the toxic and carcinogenic, polyketide synthase (PKS)-derived aflatoxins. Except for the production of aflatoxins, cyclopiazonic acid and several other metabolites the capability for metabolite production of most of these putative clusters is unknown. We investigated the regulation of expression of the PKS-non-ribosomal peptide synthetase (NRPS) containing cluster 23 and determined that it produces homologs of the known 2-pyridone leporin A. Inactivation and overexpression of a cluster 23 gene encoding a putative Zn(2)-Cys(6) transcription factor (AFLA_066900, lepE) resulted in downregulation of nine and up-regulation of 8, respectively, of the fifteen SMURF-predicted cluster 23 genes thus allowing delineation of the cluster. Overexpression of lepE (OE::lepE) resulted in transformants displaying orange-red pigmented hyphae. Mass spectral analysis of A. flavus OE::lepE extracts identified the known 2-pyridone metabolite, leporin B, as well as the previously unreported dehydroxy-precursor, leporin C. We provide strong evidence that leporin B forms a unique trimeric complex with iron, not found previously for other 2-pyridones. This iron complex demonstrated antiinsectan and antifeedant properties similar to those previously found for leporin A. The OE::lepE strain showed reduced levels of conidia and sclerotia suggesting that unscheduled leporin production affects fungal developmental programs.

Differential biochemical response of rat kidney towards low and high doses of NiCl2 as revealed by NMR spectroscopy.

Heavy metals are known for their associated nephrotoxicity and nickel is no exception. An integrated metabonomic approach, based on high-resolution (1) H NMR spectroscopy, was applied to determine the acute biochemical effects of NiCl(2) on the renal tissues of rats. Kidney homogenates from rats treated with NiCl(2) at two dose levels (4 and 20 mg kg(-1) b.w., i.p.) and those from controls were analysed using (1) H NMR spectroscopy and also assessed for antioxidant parameters at days 1, 3 and 5 post-dose. The major metabolite changes corresponding to nickel exposure were related to amino acids, osmolytes and energy metabolites. Differential responses were observed in (1) H NMR spectra with exposure to low and high doses of NiCl(2). For high doses, (1) H NMR spectral analysis revealed alterations in renal tissues, along with damage to the cortical and papillary region and depletion of renal osmolytes such as betaine, trimethyl amine oxide, myo-inositol and taurine, which persisted until day 5 post-dose. The metabolite profile of (1) H NMR spectra obtained from animals treated with lower dose of NiCl(2) initially increased as an immediate stress response and then showed signs of recovery with the passage of time. NMR spectral analysis was well corroborated with histopathological and oxidative stress results. Nickel-induced oxidative stress was observed in both groups of animals with increased levels of antioxidant parameters at initial time points, but continued to increase in the high-dose group. The present study shows a huge potential of metabonomics for mapping organ-based metabolic response during heavy metal toxicity.

Functional and phylogenetic analysis of the Aspergillus ochraceoroseus aflQ (ordA) gene ortholog.

Within the Aspergillus parasiticus and A. flavus aflatoxin (AF) biosynthetic gene cluster the aflQ (ordA) and aflP (omtA) genes encode respectively an oxidoreductase and methyltransferase. These genes are required for the final steps in the conversion of sterigmatocystin (ST) to aflatoxin B(1) (AFB(1)). Aspergillus nidulans harbors a gene cluster that produces ST, as the aflQ and aflP orthologs are either non-functional or absent in the genome. Aspergillus ochraceoroseus produces both AF and ST, and it harbors an AF/ST biosynthetic gene cluster that is organized much like the A. nidulans ST cluster. The A. ochraceoroseus cluster also does not contain aflQ or aflP orthologs. However the ability of A. ochraceoroseus to produce AF would indicate that functional aflQ and aflP orthologs are present within the genome. Utilizing degenerate primers based on conserved regions of the A. flavus aflQ gene and an A. nidulans gene demonstrating the highest level of homology to aflQ, a putative aflQ ortholog was PCR amplified from A. ochraceoroseus genomic DNA. The A. ochraceoroseus aflQ ortholog demonstrated 57% amino acid identity to A. flavus AflQ. Transformation of an O-methylsterigmatocystin (OMST)-accumulating A. parasiticus aflQ mutant with the putative A. ochraceoroseus aflQ gene restored AF production. Although the aflQ gene does not reside in the AF/ST cluster it appears to be regulated in a manner similar to other A. ochraceoroseus AF/ST cluster genes. Phylogenetic analysis of AflQ and AflQ-like proteins from a number of ST- and AF-producing Aspergilli indicates that A. ochraceoroseus might be ancestral to A. nidulans and A. flavus.

Development of sexual structures influences metabolomic and transcriptomic profiles in Aspergillus flavus.

Sclerotium (female) fertility, the ability of a strain to produce ascocarps, influences internal morphological changes during sexual reproduction in Aspergillus flavus. Although sclerotial morphogenesis has been linked to secondary metabolite (SM) biosynthesis, metabolic and transcriptomic changes within A. flavus sclerotia during sexual development are not known. Successful mating between compatible strains may result in relatively high or low numbers of ascocarps being produced. Sclerotia from a high fertility cross (Hi-Fert-Mated), a low fertility cross (Lo-Fert-Mated), unmated strains (Hi-Fert-Unmated and Lo-Fert-Unmated) were harvested immediately after crosses were made and every two weeks until 8 weeks of incubation, then subjected to targeted metabolomics (n = 106) and transcriptomics analyses (n = 80). Aflatoxin B1 production varied between Hi-Fert-Mated and Hi-Fert-Unmated sclerotia, while it remained low or was undetected in Lo-Fert-Mated and Lo-Fert-Unmated sclerotia. Profiling of 14 SMs showed elevated production of an aflavazole analog, an aflavinine isomer, and hydroxyaflavinine in Hi-Fert-Mated sclerotia at 4 to 8 weeks. Similarly, genes ayg1, hxtA, MAT1, asd-3, preA and preB, and genes in uncharacterized SM gene clusters 30 and 44 showed increased expression in Hi-Fert-Mated sclerotia at these time points. These results broaden our knowledge of the biochemical and transcriptional processes during sexual development in A. flavus.