The first two decades of vitamin E.

This presentation reviews highlights of the first 20 years (1922-1942) of vitamin E. It begins with background information leading to identification of an antisterility factor for rats of both sexes and its acceptance into the vitamin family as vitamin E (1925). Research of the next 12 years revealed a multiplicity of deficiency manifestations: embryonic mortality, testis degeneration, encephalomalacia and exudative diathesis in the chick, and nutritional muscular dystrophy in avian and mammalian species. Toward the close of this period came the isolation of vitamin E from natural sources, determination of its empirical formula, and introduction of the designation alpha-tocopherol for vitamin E (1936). Within the next two years the structural formula of alpha-tocopherol was elucidated, its chemical synthesis accomplished, and its production from natural plant oils by molecular distillation was well established. The existence of other tocopherols with lesser degrees of biological activity became recognized. Also, the concurrent development of a chemical method for determining the vitamin E content of alpha-tocopherol in foods, body tissues and body fluids, which replaced the very laborious bioassay procedure, greatly facilitated later advances in knowledge of the distribution and nature of vitamin E.     

Gene therapy: the first two decades and the current state-of-the-art

The concept of gene therapy was envisioned soon after the emergence of restriction endonucleases and subcloning of mammalian genes in phage and plasmids. Over the ensuing decades, vectors were developed, including nonviral methods, integrating virus vectors (gammaretrovirus and lentivirus), and non-integrating virus vectors (adenovirus, adeno-associated virus, and herpes simplex virus vectors). Preclinical data demonstrated potential efficacy in a broad range of animal models of human diseases, but clinical efficacy in humans remained elusive in most cases, even after decades of experience in over 1000 trials. Adverse effects from gene therapy have been observed in some cases, often because of viral vectors retaining some of the pathogenic potential of the viruses upon which they are based. Later generation vectors have been developed in which the safety and/or the efficiency of gene transfer has been improved. Most recently this work has involved alterations of vector envelope or capsid proteins either by insertion of ligands to target specific receptors or by directed evolution. The disease targets for gene therapy are multiple, but the most promising data have come from monogenic disorders. As the number of potential targets for gene therapy continues to increase, and a substantial number of trials continue with both the standard and the later generation vector systems, it is hoped that a therapeutic niche for gene therapy will emerge in the coming decades.     

Interactive learning: Lessons from two hybrids over two decades

The initial yeast two‐hybrid experiment – published in 1989 – described an approach to detecting protein–protein interactions that has flourished over the last two decades, leading to the assembly of large‐scale data sets of these interactions. Yet the yeast assay originated because of the laboratory's interests in technology development, not because of its need to identify partners of any protein then under study. In addition to such motivating forces, other features of the process of originating a technology can be revealed by considering the lessons of the two‐hybrid approach. These include the value of timeliness in a method's development, the willingness of an investigator to try experimental approaches that prove fruitless, the ability of biological macromolecules to display surprising attributes, the benefits of a community expending efforts to expand the uses of a technology platform, and the role of scientific training of those who work in technology.     

The first family of cell-cell fusion

Our understanding of the developmentally critical process of cell-cell fusion has been greatly advanced by the identification of the first family of cell-cell fusion proteins. Together, the two founding members of the FF family execute the majority of cell-cell fusion events in C. elegans.     

Anguillicolosis: dynamics of the infection over two decades

The dynamics of the infection of the European eel Anguilla anguilla L. by the Asian nematode Anguillicola crassus Kuwahara, Niimi and Itagaki, 1974 (i.e. anguillicolosis) was monitored over 2 decades in an oligohaline canal in southern France (Camargue, Mediterranean coast). Since the first mention of the parasite in this canal in 1985, which was also the first record in France, prevalence of pre-adult and adult forms has risen from 32 to 73%. However, during the last 7 yr (1997 to 2003), prevalence seems to have stabilized around values of 60 to 70% and parasite load, though inter-annual variation is substantial, shows no sign of increase (intensity for the last 5 yr: min. = 3.70, max. = 9.66, mean = 6.01). Our results thus confirm the dynamic pattern observed elsewhere in Europe, i.e. a rapid spread following the introduction of the parasite in a water system and then stabilization around ceiling levels. We review possible mechanisms that may explain such a leveling off in the infection spread. We particularly document the possibility that repetitive infections may render the infected organ, i.e. the swimbladder, unsuitable for further A. crassus establishment. In support of this hypothesis, we showed that the infection rate is lower among eels with severely damaged swimbladders.     

Animal pharming,two decades on

Since its inception 20 years ago, the animal pharming industry has promoted transgenic animals as a cost-effective method of biopharmaceutical production. However, it took until 2006 for the first therapeutic product to gain regulatory approval. This was an important milestone, but scepticism still abounds. Can pharming regain investor confidence, and will society accept transgenic livestock as a production method? There is some cause for optimism, biopharmaceuticals are a large, expanding market and animal pharming has already made considerable strides. A novel production platform has been established, groundbreaking technologies developed, a necessary regulatory framework put in place. Nevertheless, despite cost advantages, pharming has become a niche production method and its long term success may depend on products unique to transgenic animals.     

Alkaline ceramidase 3 deficiency aggravates colitis and colitis-associated tumorigenesis in mice by hyperactivating the innate immune system

Increasing studies suggest that ceramides differing in acyl chain length and/or degree of unsaturation have distinct roles in mediating biological responses. However, still much remains unclear about regulation and role of distinct ceramide species in the immune response. Here, we demonstrate that alkaline ceramidase 3 (Acer3) mediates the immune response by regulating the levels of C_18:1-ceramide in cells of the innate immune system and that Acer3 deficiency aggravates colitis in a murine model by augmenting the expression of pro-inflammatory cytokines in myeloid and colonic epithelial cells (CECs). According to the NCBI Gene Expression Omnibus (GEO) database, ACER3 is downregulated in immune cells in response to lipopolysaccharides (LPS), a potent inducer of the innate immune response. Consistent with these data, we demonstrated that LPS downregulated both Acer3 mRNA levels and its enzymatic activity while elevating C_18:1-ceramide, a substrate of Acer3, in murine immune cells or CECs. Knocking out Acer3 enhanced the elevation of C_18:1-ceramide and the expression of pro-inflammatory cytokines in immune cells and CECs in response to LPS challenge. Similar to Acer3 knockout, treatment with C_18:1-ceramide, but not C_18:0-ceramide, potentiated LPS-induced expression of pro-inflammatory cytokines in immune cells. In the mouse model of dextran sulfate sodium-induced colitis, Acer3 deficiency augmented colitis-associated elevation of colonic C_18:1-ceramide and pro-inflammatory cytokines. Acer3 deficiency aggravated diarrhea, rectal bleeding, weight loss and mortality. Pathological analyses revealed that Acer3 deficiency augmented colonic shortening, immune cell infiltration, colonic epithelial damage and systemic inflammation. Acer3 deficiency also aggravated colonic dysplasia in a mouse model of colitis-associated colorectal cancer. Taken together, these results suggest that Acer3 has an important anti-inflammatory role by suppressing cellular or tissue C_18:1-ceramide, a potent pro-inflammatory bioactive lipid and that dysregulation of ACER3 and C_18:1-ceramide may contribute to the pathogenesis of inflammatory diseases including cancer.Ceramides are the central lipid in the metabolic network of sphingolipids, and are generated through the de novo, catabolic and salvage pathways.¹ In the de novo pathway, ceramides are synthesized through multiple steps catalyzed sequentially by serine palmitoyltransferase (SPT), keto-dihydrosphingosine reductase, (dihydro)ceramide synthases (CerSs) and dihydroceramide desaturases. In the catabolic pathways, ceramides are derived from the hydrolysis of sphingomyelins by sphingomyelinases (SMases) or the hydrolysis of glycosphingolipids. In the salvage pathway, ceramides are synthesized from sphingosine (SPH) and fatty acyl-CoA by CerSs. As CerSs (CerS1-6) have distinct specificity toward acyl-CoA chain length and degree of unsaturation, ceramides with various acyl-chains are found in mammalian cells. Upon generation, ceramides can be hydrolyzed by five ceramidases encoded by five distinct genes (ASAH1, ASAH2, ACER1, ACER2 and ACER3). These ceramidases vary in pH optimum for catalytic activity, tissue distribution, cellular localization and substrate specificity,² allowing for regulation of specific ceramides in a cell- or tissue-specific manner.Recent studies have implicated ceramides in regulating the innate immune response. Sakata et al.³ demonstrated that lipopolysaccharides (LPS), a potent inducer of the innate immune response, increases C₁₆-ceramide by activating acid SMase and that inhibition of SMase attenuates LPS-induced production of pro-inflammatory cytokines in THP-1 macrophages. Andreyev et al.⁴ found that ceramides are increased by Toll-like receptor 4 (TLR4)-specific LPS in RAW 264.7 macrophages. Schilling et al.⁵ revealed that LPS and palmitic acid synergistically increase C₁₆-ceramide in primary mouse peritoneal macrophages (PMs) by activating de novo biosynthesis of ceramides and that inhibiting the C₁₆-ceramide increase attenuates LPS-induced production of TNF-α and IL-1β in PMs. A recent study found that LPS increases ceramides in Raw 264.7 macrophages through nuclear factor kappa B (NF-κB)-dependent upregulation of SPT long chain base subunit 2 Sptlc2, a regulator of SPT.⁶ These results suggest that ceramides mediate the immune response in part by enhancing the production of pro-inflammatory cytokines in innate immune cells.Emerging evidence suggests that dysregulation in the innate immune response in inflammatory bowel disease (IBD) contributes to the pathogenesis of the disease.⁷ Consistent with the role of ceramides in potentiating the innate immune response, several studies found that ceramides may have a role in the pathogenesis of IBD. Sakata et al.³ demonstrated that blocking the generation of ceramides with the SMase inhibitor hinders mouse colitis. Fischbeck et al.⁸ showed that increasing ceramides in the gut by supplying mice with dietary sphingomyelins, a precursor of ceramides, aggravates mouse colitis. These results suggest that increased levels of ceramides may contribute to the pathogenesis of IBD.Although the role of ceramides and their generating enzymes in the innate immune response have been well studied, much remains unclear about the role of ceramidases involved in the catabolism of ceramides in this biological response. In this study, we investigated the role of alkaline ceramidase 3 (ACER3)/Acer3 and its substrates in immune response. We demonstrated that Acer3 is downregulated, whereas its substrate, C_18:1-ceramide, is upregulated in murine immune cells and colonic epithelial cells (CECs) during the innate immune response to LPS. Using Acer3 null mice (Acer3^−/−) and their wild-type (Acer3^+/+) littermates, we further discovered that the inverse regulation of Acer3 and C_18:1-ceramide potentiates LPS-induced production of pro-inflammatory cytokines in innate immune cells. More importantly, we found that Acer3 deficiency aggravates dextran sulfate sodium (DSS)-induced colitis and colitis-associated colorectal cancer (CAC) in a murine model. These findings indicate that Acer3/ACER3 and C_18:1-ceramide are novel modulators in the innate immune response and that their dysregulation may contribute to the pathogenesis of inflammatory diseases.     

Plasmid rolling-circle replication: highlights of two decades of research

This review provides a historical perspective of the major findings that contributed to our current understanding of plasmid rolling-circle (RC) replication. Rolling-circle-replicating (RCR) plasmids were discovered approximately 20 years ago. The first of the RCR plasmids to be identified were native to Gram-positive bacteria, but later such plasmids were also identified in Gram-negative bacteria and in archaea. Further studies revealed mechanistic similarities in the replication of RCR plasmids and the single-stranded DNA bacteriophages of Escherichia coli, although there were important differences as well. Three important elements, a gene encoding the initiator protein, the double strand origin, and the single strand origin, are contained in all RCR plasmids. The initiator proteins typically contain a domain involved in their sequence-specific binding to the double strand origin and a domain that nicks within the double strand origin and generates the primer for DNA replication. The double strand origins include the start-site of leading strand synthesis and contain sequences that are bound and nicked by the initiator proteins. The single strand origins are required for synthesis of the lagging strand of RCR plasmids. The single strand origins are non-coding regions that are strand-specific, and contain extensive secondary structures. This minireview will highlight the major findings in the study of plasmid RC replication over the past twenty years. Regulation of replication of RCR plasmids will not be included since it is the subject of another review.     

The Severn Estuary's changing wader populations during the last two decades

Many of the changes in population size which have occurred in the wading birds of the Severn Estuary reflect changes that have taken place in Britain as a whole. An important exception is the dunlin, which has maintained its numbers in the Severn despite undergoing a significant decline nationally. The fluctuations of several species show evidence of the effects of the hard winters of 1979, 1982 and 1986, the dry conditions of 1976 and perhaps of good breeding seasons in the high Arctic. Bridgwater Bay has declined in importance for nearly all species, with many individuals appearing to have switched to the Rumney on the northern shore.     

The reverse activity of human acid ceramidase

An overexpression system was recently developed to produce and purify recombinant, human acid ceramidase. In addition to ceramide hydrolysis, the purified enzyme was able to catalyze ceramide synthesis using [14C]lauric acid and sphingosine as substrates. Herein we report detailed characterization of this acid ceramidase-associated "reverse activity" and provide evidence that this reaction occurs in situ as well as in vitro. The pH optimum of the reverse reaction was approximately 5.5, as compared with approximately 4.5 for the hydrolysis reaction. Non-ionic detergents and zinc cations inhibited the activity, whereas most other cations were stimulatory. Of note, sphingomyelin also was very inhibitory toward this reaction, whereas the anionic lipids, phosphatidic acid and phosphatidylserine, were stimulatory. Of various sphingosine stereoisomers tested in the reverse reaction, only the natural, D-erythro form could efficiently serve as a substrate. Using D-erythro-sphingosine and lauric acid as substrates, the reaction followed normal Michaelis-Menten kinetics. The Km and Vmax values toward sphingosine were 23.75 microM and 208.3 pmol/microg/h, respectively, whereas for lauric acid they were 73.76 microM and 232.5 pmol/microg/h, respectively. Importantly, the reverse activity was reduced in cell lysates from a Farber disease patient to the same extent as the acid ceramidase activity. Furthermore, when 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) (NBD)-conjugated lauric acid and sphingosine were added to cultured lymphoblasts from a Farber disease patient in the presence of fumonisin B (1), the conversion to NBD-ceramide was reduced approximately 30% when compared with normal cells. These data provide important new information on human acid ceramidase and further document its central role in sphingolipid metabolism.     

The tiger mosquito in Lebanon two decades after its introduction: A growing health concern

The tiger mosquito was introduced to the Eastern region of the Mediterranean basin more than twenty years ago. In Lebanon, it was first observed in 2002 in a limited number of locations mainly from the coastal area of the country. In the absence of national entomological control program, this invasive mosquito became an established species and is now considered in many localities, a source of nuisance because of its human biting behavior. Several entomological surveys were conducted to monitor the geographic spread and the seasonal dynamics of Aedes albopictus by collecting adult stages and by monitoring oviposition activity. Moreover, its susceptibility to the common groups of insecticides was assessed using WHO standard bioassays. Previous vector competence studies revealed that local strains were able to transmit Chikungunya and Dengue viruses. Due to the increased risk of Zika virus introduction in the country, we determined the competence of local populations to transmit this virus. Mapping results showed that Ae. albopictus is mainly spread in the relatively humid western versant of the Mount Lebanon chain reaching 1000m altitude, while it is absent from arid and semi-arid inland areas. Besides, this mosquito is active during 32 weeks from spring till the end of autumn. Local strains of the tiger mosquito are susceptible to pyrethroids and carbamates but resistant to organophosphates and organochlorines. They showed ability to transmit Zika virus; however, only 9% of females were capable to excrete the virus in their saliva at day 28 post infection. Current and previous observations highlight the need to establish a surveillance system in order to control this mosquito and monitor the potential introduction of related diseases.     

Survival in treated hypertension: follow up study after two decades

Objective: To compare survival and cause specific mortality in hypertensive men with non-hypertensive men derived from the same random population, and to study mortality and morbidity from cardiovascular diseases in the hypertensive men in relation to effects on cardiovascular risk factors during 22-23 years of follow up. Design: Prospective, population based observational study. Subjects and methods: 686 hypertensive men aged 47-55 at screening compared with 6810 non-hypertensive men. The hypertensive men were having stepped care treatment with either β adrenergic blocking drugs, thiazide diuretics, or combination treatment. Mortality, morbidity, and adverse effects were registered at yearly examinations and from death certificates. Main outcome measures: All cause mortality and cause specific mortality. Results: Treated hypertensive men had significantly impaired probability of total survival as well as survival from coronary heart disease and stroke. All cause mortality as well as coronary heart disease and stroke mortality were very similar in hypertensive men and normotensive men during the first decade, but increased steadily thereafter despite continuous good blood pressure control. Smoking, signs of target organ damage, and high serum cholesterol levels, but not blood pressure at screening, were significantly related to the incidence of coronary heart disease during follow up. In time dependent Cox’s regression analysis, the incidence of coronary heart disease was significantly related only to serum cholesterol concentrations in the study. Cancer mortality was almost similar in treated hypertensive men (61/686, 8.9%) and non-hypertensive men (732/6810, 10.8%). Conclusion: Treated hypertensive men had impaired survival and increased mortality from cardiovascular disease compared with non-hypertensive men of similar age. These differences were observed during the second decade of follow up. During an observation period of 22-23 years—about 15 000 patient years—hypertensive men receiving diuretics and β blockers had no increased risk of cancer or non-cardiovascular disease.

Key messages

• Hypertension is a prevalent (10-20%) and important risk factor for cardiovascular disease.
• In controlled trials over 3-5 years drug treatment for hypertension prevents these complications, but little is known about long term prognosis
• During 20-22 years treated hypertensive men had a significantly increased mortality, especially from coronary heart disease, compared with non-hypertensive men from the same population
• The high incidence of myocardial infarction was related to organ damage, smoking, and cholesterol at the time of entry to the study, and to achieved serum cholesterol concentrations during follow up
• The poor prognosis for mortality from coronary heart disease is dependent upon strict monitoring of serum cholesterol concentrations

     

The search for human pheromones: the lost decades and the necessity of returning to first principles

As humans are mammals, it is possible, perhaps even probable, that we have pheromones. However, there is no robust bioassay-led evidence for the widely published claims that four steroid molecules are human pheromones: androstenone, androstenol, androstadienone and estratetraenol. In the absence of sound reasons to test the molecules, positive results in studies need to be treated with scepticism as these are highly likely to be false positives. Common problems include small sample sizes, an overestimate of effect size (as no effect can be expected), positive publication bias and lack of replication. Instead, if we are to find human pheromones, we need to treat ourselves as if we were a newly discovered mammal, and use the rigorous methods already proven successful in pheromone research on other species. Establishing a pheromone relies on demonstration of an odour-mediated behavioural or physiological response, identification and synthesis of the bioactive molecule(s), followed by bioassay confirmation of activity. Likely sources include our sebaceous glands. Comparison of secretions from adult and pre-pubertal humans may highlight potential molecules involved in sexual behaviour. One of the most promising human pheromone leads is a nipple secretion from the areola glands produced by all lactating mothers, which stimulates suckling by any baby not just their own.     

The family Agaricaceae: phylogenies and two new white-spored genera

A well resolved phylogeny of the Agaricaceae based on partial rpb2 sequences is presented from a wide geographic and systematic sampling of the family and compared to phylogenies based on nrLSU and tef1 sequences. A smaller dataset of the family focusing on the Agaricus clade of nrITS sequences and a combined dataset were used to determine the position of several white-spored taxa from northern Thailand. Two new genera are described from Thailand. Coniolepiota accommodates Lepiota spongodes, a gray-lilac-purple floccose white-spored species with a wide distribution in tropical Asia; Eriocybe has a white wooly felt-like covering of pileus and stipe, white spores and is described with one new species E. chionea, so far known only from northern Thailand. These new genera are closely related to three genera with colored spores (viz. Agaricus, Heinemannomyces and Clarkeinda) and not to other white-spored taxa.     

The impact of taxon sampling on phylogenetic inference: a review of two decades of controversy

Over the past two decades, there has been a long-standing debate about the impact of taxon sampling on phylogenetic inference. Studies have been based on both real and simulated data sets, within actual and theoretical contexts, and using different inference methods, to study the impact of taxon sampling. In some cases, conflicting conclusions have been drawn for the same data set. The main questions explored in studies to date have been about the effects of using sparse data, adding new taxa, including more characters from genome sequences and using different (or concatenated) locus regions. These questions can be reduced to more fundamental ones about the assessment of data quality and the design guidelines of taxon sampling in phylogenetic inference experiments. This review summarizes progress to date in understanding the impact of taxon sampling on the accuracy of phylogenetic analysis.