Targeting Chikungunya Virus Replication by Benzoannulene Inhibitors.

A benzo[6]annulene, 4-(tert-butyl)-N-(3-methoxy-5,6,7,8-tetrahydronaphthalen-2-yl) benzamide (1a), was identified as an inhibitor against Chikungunya virus (CHIKV) with antiviral activity EC90 = 1.45 µM and viral titer reduction (VTR) of 2.5 log at 10 µM with no observed cytotoxicity (CC50 = 169 µM) in normal human dermal fibroblast cells. Chemistry efforts to improve potency, efficacy, and drug-like properties of 1a resulted in a novel lead compound 8q, which possessed excellent cellular antiviral activity (EC90 = 270 nM and VTR of 4.5 log at 10 µM) and improved liver microsomal stability. CHIKV resistance to an analog of 1a, compound 1c, tracked to a mutation in the nsP3 macrodomain. Further mechanism of action studies showed compounds working through inhibition of human dihydroorotate dehydrogenase in addition to CHIKV nsP3 macrodomain. Moderate efficacy was observed in an in vivo CHIKV challenge mouse model for compound 8q as viral replication was rescued from the pyrimidine salvage pathway.

Cellular stress responses to chronic heat shock and shell damage in temperate Mya truncata.

Acclimation, via phenotypic flexibility, is a potential means for a fast response to climate change. Understanding the molecular mechanisms underpinning phenotypic flexibility can provide a fine-scale cellular understanding of how organisms acclimate. In the last 30 years, Mya truncata populations around the UK have faced an average increase in sea surface temperature of 0.7 °C and further warming of between 1.5 and 4 °C, in all marine regions adjacent to the UK, is predicted by the end of the century. Hence, data are required on the ability of M. truncata to acclimate to physiological stresses, and most notably, chronic increases in temperature. Animals in the present study were exposed to chronic heat-stress for 2 months prior to shell damage and subsequently, only 3, out of 20 damaged individuals, were able to repair their shells within 2 weeks. Differentially expressed genes (between control and damaged animals) were functionally enriched with processes relating to cellular stress, the immune response and biomineralisation. Comparative transcriptomics highlighted genes, and more broadly molecular mechanisms, that are likely to be pivotal in this lack of acclimation. This study demonstrates that discovery-led transcriptomic profiling of animals during stress-response experiments can shed light on the complexity of biological processes and changes within organisms that can be more difficult to detect at higher levels of biological organisation.

Unusual tissue distribution of carcinin, an antibacterial crustin, in the crab, Carcinus maenas, reveals its multi-functionality.

Crustins are whey acidic four-disulphide core (WFDSC) domain-containing proteins in decapods that are widely regarded as antimicrobial agents that contribute to host defence. Whilst there have been many analyses of crustin gene expression in tissues, few studies have been made of the distribution of the natural proteins. Here we report an immunostaining investigation of carcinin, a native crustin from Carcinus maenas, in the body organs. The results show that the protein is largely confined to the haemocytes with only a weak signal detected in the heart, hepatopancreas and midgut caecum where it is restricted to the outer surfaces. Importantly, carcinin was seen to be deposited by the haemocytes on these surfaces. Higher levels of staining were detected in the gonads with carcinin particularly abundant in the capsule of ovary as well as some oocytes. Conspicuous staining was further evident in the cuticle of the eyestalk peduncles. Ablation of the eyestalks resulted in a reduction of carcinin in the maturing ovary with the mature eggs rarely displaying a strong signal for the protein. Interestingly, the degree of carcinin also strongly increased in the healing peduncle, indicating that the protein may be associated with wounding, cell damage and/or tissue regeneration.

WIND TURBINES CAUSE CHRONIC STRESS IN BADGERS (MELES MELES) IN GREAT BRITAIN.

A paucity of data exists with which to assess the effects of wind turbines noise on terrestrial wildlife, despite growing concern about the impact of infrasound from wind farms on human health and well-being. In 2013, we assessed whether the presence of turbines in Great Britain impacted the stress levels of badgers ( Meles meles ) in nearby setts. Hair cortisol levels were used to determine if the badgers were physiologically stressed. Hair of badgers living <1 km from a wind farm had a 264% higher cortisol level than badgers >10 km from a wind farm. This demonstrates that affected badgers suffer from enhanced hypothalamo-pituitary-adrenal activity and are physiologically stressed. No differences were found between the cortisol levels of badgers living near wind farms operational since 2009 and 2012, indicating that the animals do not become habituated to turbine disturbance. Cortisol levels in the affected badgers did not vary in relation to the distance from turbines within 1 km, wind farm annual power output, or number of turbines. We suggest that the higher cortisol levels in affected badgers is caused by the turbines' sound and that these high levels may affect badgers' immune systems, which could result in increased risk of infection and disease in the badger population.

Antimicrobial proteins: From old proteins, new tricks.

This review describes the main types of antimicrobial peptides (AMPs) synthesised by crustaceans, primarily those identified in shrimp, crayfish, crab and lobster. It includes an overview of their range of microbicidal activities and the current landscape of our understanding of their gene expression patterns in different body tissues. It further summarises how their expression might change following various types of immune challenges. The review further considers proteins or protein fragments from crustaceans that have antimicrobial properties but are more usually associated with other biological functions, or are derived from such proteins. It discusses how these unconventional AMPs might be generated at, or delivered to, sites of infection and how they might contribute to crustacean host defence in vivo. It also highlights recent work that is starting to reveal the extent of multi-functionality displayed by some decapod AMPs, particularly their participation in other aspects of host protection. Examples of such activities include proteinase inhibition, phagocytosis, antiviral activity and haematopoiesis.

Disk Diffusion Assay to Assess the Antimicrobial Activity of Marine Algal Extracts.

Marine algae are a relatively untapped source of bioactive natural products, including those with antimicrobial activities. The ability to assess the antimicrobial activity of cell extracts derived from algal cultures is vital to identifying species that may produce useful novel antibiotics. One assay that is used widely for this purpose is the disk diffusion assay due to its simplicity, rapidity, and low cost. Moreover, this assay gives output data that are easy to interpret and can be used to screen many samples at once irrespective of the solvent used during preparation. In this chapter, a step-by-step protocol for performing a disk diffusion assay is described. The assay is particularly well suited to testing algal cell extracts and fractions resulting from separation through bioassay-guided approaches.

Invertebrate extracellular phagocyte traps show that chromatin is an ancient defence weapon.

Controlled release of chromatin from the nuclei of inflammatory cells is a process that entraps and kills microorganisms in the extracellular environment. Now termed ETosis, it is important for innate immunity in vertebrates. Paradoxically, however, in mammals, it can also contribute to certain pathologies. Here we show that ETosis occurs in several invertebrate species, including, remarkably, an acoelomate. Our findings reveal that the phenomenon is primordial and predates the evolution of the coelom. In invertebrates, the released chromatin participates in defence not only by ensnaring microorganisms and externalizing antibacterial histones together with other haemocyte-derived defence factors, but crucially, also provides the scaffold on which intact haemocytes assemble during encapsulation; a response that sequesters and kills potential pathogens infecting the body cavity. This insight into the early origin of ETosis identifies it as a very ancient process that helps explain some of its detrimental effects in mammals.

Changes in seasonal expression patterns of ecdysone receptor, retinoid X receptor and an A-type allatostatin in the copepod, Calanus finmarchicus, in a sea loch environment: an investigation of possible mediators of diapause.

The marine copepod, Calanus finmarchicus, is a crucial component of the pelagic food web in the North Atlantic and peripheral seas where it is a major player in biogeochemical cycles and the productivity of commercially important fisheries. A key stage in its life cycle is the emergence of the pre-adult, copepodite developmental stage five (CV) from a period of overwintering dormancy, known as diapause. As is the case in many insect species, diapause is also likely to be under endocrine control in C. finmarchicus. To investigate the hormonal regulation of diapause behaviour of stage CV C. finmarchicus, the expression of three key genes: ecdysone receptor (EcR), retinoid X receptor (RXR) and an A-type allatostatin (A-type AST), were measured in specimens collected at monthly intervals from Loch Etive, a ca. 150m deep sea loch on the west coast of Scotland, between June 2006 and May 2007. The full length RXR gene was cloned and sequenced from C. finmarchicus, and was found to share 49-53% total identity with equivalent genes encoding proteins from other crustaceans, and >80% identity in the DNA binding domain with other crustaceans, insects and vertebrates. EcR expression was least in December when the animals are expected to be in diapause, but began to increase in January, when the animals were terminating diapause. Concomittant with the rise in EcR in January was low expression of A-type AST and high expression of RXR.

Synthesis and pharmacology of 1-alkyl-3-(1-naphthoyl)indoles: steric and electronic effects of 4- and 8-halogenated naphthoyl substituents.

To develop SAR at both the cannabinoid CB(1) and CB(2) receptors for 3-(1-naphthoyl)indoles bearing moderately electron withdrawing substituents at C-4 of the naphthoyl moiety, 1-propyl and 1-pentyl-3-(4-fluoro, chloro, bromo and iodo-1-naphthoyl) derivatives were prepared. To study the steric and electronic effects of substituents at the 8-position of the naphthoyl group, the 3-(4-chloro, bromo and iodo-1-naphthoyl)indoles were also synthesized. The affinities of both groups of compounds for the CB(1) and CB(2) receptors were determined and several of them were evaluated in vivo in the mouse. The effects of these substituents on receptor affinities and in vivo activity are discussed and structure-activity relationships are presented. Although many of these compounds are selective for the CB(2) receptor, only three JWH-423, 1-propyl-3-(4-iodo-1-naphthoyl)indole, JWH-422, 2-methyl-1-propyl-3-(4-iodo-1-naphthoyl)indole, the 2-methyl analog of JWH-423 and JWH-417, 1-pentyl-3-(8-iodo-1-naphthoyl)indole, possess the desirable combination of low CB(1) affinity and good CB(2) affinity.

Phylogeny of whey acidic protein (WAP) four-disulfide core proteins and their role in lower vertebrates and invertebrates.

Proteins containing WAP (whey acidic protein) domains with a characteristic WFDC (WAP four-disulfide core) occur not only in mammals (including marsupials and monotremes) but also in birds, reptiles, amphibians and fish. In addition, they are present in numerous invertebrates, from cnidarians to urochordates. Many of those from non-mammalian groups are poorly understood with respect to function or phylogeny. Those well characterized so far are waprins from snakes, perlwapins from bivalves and crustins from decapod crustaceans. Waprins are venom proteins with a single WAP domain at the C-terminus. They display antimicrobial, rather than proteinase inhibitory, activities. Perlwapins, in contrast, possess three WAP domains at the C-terminus and are expressed in the shell nacre of abalones. They participate in shell formation by inhibiting the growth of calcium crystals in the shell. The crustin group is the largest of all WFDC-containing proteins in invertebrates with the vast majority being highly expressed in the haemocytes. Most have a single WAP domain at the C-terminus. The presence and type of the domains between the signal sequence and the C-terminus WAP domain separate the different crustin types. Most of the Type I and II crustins are antimicrobial towards Gram-positive bacteria, whereas the Type III crustins tend to display protease inhibition. Expression studies show that at least some crustins have other important biological effects, as levels change with physiological stress, wound repair, tissue regeneration or ecdysis. Thus WAP domains are widely distributed and highly conserved, serving in diverse physiological processes (proteinase inhibition, bacterial killing or inhibition of calcium transport).

Isolation and in vitro characterisation of prohaemocytes from the spider crab, Hyas araneus (L.).

A population of small, mostly undifferentiated, haemocytes were identified and enriched from the circulation of the spider crab, H. araneus, using a two-step density gradient separation procedure. Typically, these cells are spherical, ca. 8-12 µm diameter and have a high nucleus:cytoplasm ratio. Their number in the circulation increases significantly 24 h after a state of haemocytopenia has been created by withdrawal of 2 mL of haemolymph. The rise in the number of these cells at this time is consistent with a left shift phenomenon. A two-step separation procedure was developed to generate enriched populations of these small cells from the haemolymph and in vitro assays revealed that ca. 47% are BrdU-positive in vitro. By contrast BrdU uptake was not observed in the hyaline, semigranular or granular cells. The proliferative ability of the small cells, coupled with their close morphological resemblance to immature haemocytes reported from the haematopoietic tissue of other decapod species, leads us to conclude that these cells are prohaemocytes.

Conventional and unconventional antimicrobials from fish, marine invertebrates and micro-algae.

All eukaryotic organisms, single-celled or multi-cellular, produce a diverse array of natural anti-infective agents that, in addition to conventional antimicrobial peptides, also include proteins and other molecules often not regarded as part of the innate defences. Examples range from histones, fatty acids, and other structural components of cells to pigments and regulatory proteins. These probably represent very ancient defence factors that have been re-used in new ways during evolution. This review discusses the nature, biological role in host protection and potential biotechnological uses of some of these compounds, focusing on those from fish, marine invertebrates and marine micro-algae.

Expression of antimicrobial peptides from Hyas araneus haemocytes following bacterial challenge in vitro.

Circulating haemocytes play major roles in the host defense reactions of decapods, including the synthesis and release of antimicrobial peptides (AMPs). Unlike the AMPs from insects, those in decapods are constitutively expressed. This study aims to establish primary cell cultures of the three main haemocyte types in Hyas araneus haemocytes, and to measure the in vitro expression of AMP genes in the cells following microbial challenge. The haemocyte populations were separated on Percoll gradients and cultured in modified L-15 medium. Expression analysis by real-time RT-PCR showed that the granular cells are the main producers of crustin, hyastatin and arasin 1 AMPs, but the hyaline cells and semigranular cells also show some expression of these genes. Incubating the cell populations with Aerococcus viridans var. homari (a Gram-positive bacterium) or Listonella anguillarum (a Gram-negative pathogen) provoked no dramatic changes in the gene expression of any of the AMP, and although there was a small (single doubling) significant increase in expression of the crustin gene in granular cells 24h after exposure to L. anguillarum, it is unclear if this is biologically relevant under in vitro conditions. The results presented in this study are in accordance with several in vivo studies.

Antibacterial free fatty acids: activities, mechanisms of action and biotechnological potential.

Amongst the diverse and potent biological activities of free fatty acids (FFAs) is the ability to kill or inhibit the growth of bacteria. The antibacterial properties of FFAs are used by many organisms to defend against parasitic or pathogenic bacteria. Whilst their antibacterial mode of action is still poorly understood, the prime target of FFA action is the cell membrane, where FFAs disrupt the electron transport chain and oxidative phosphorylation. Besides interfering with cellular energy production, FFA action may also result from the inhibition of enzyme activity, impairment of nutrient uptake, generation of peroxidation and auto-oxidation degradation products or direct lysis of bacterial cells. Their broad spectrum of activity, non-specific mode of action and safety makes them attractive as antibacterial agents for various applications in medicine, agriculture and food preservation, especially where the use of conventional antibiotics is undesirable or prohibited. Moreover, the evolution of inducible FFA-resistant phenotypes is less problematic than with conventional antibiotics. The potential for commercial or biomedical exploitation of antibacterial FFAs, especially for those from natural sources, is discussed.

Ethical and effective ethnographic research methods: a case study with afghan refugees in California.

SCHOLARLY STUDIES OF REFUGEES and other vulnerable populations carry special ethical concerns. In this invited case study of Afghan refugees in Fremont, California, I provide illustrations and recommendations of ethical research methods with refugees. I also compare and contrast some ethical issues in the U.S. with issues in Thailand. The qualitative, ethnographic methods I report here demonstrate how to conduct culturally sensitive investigations by ethically approaching gatekeepers and other community members to preserve autonomy, ensure confidentiality, build trust, and improve the accuracy of interpretations and results. Six groups at risk for being marginalized in multiple ways within refugee populations are described. Ten best practices are recommended for ethically acquiring an in-depth understanding of the refugees, their community, and appropriate research methods.

A fatty acid from the diatom Phaeodactylum tricornutum is antibacterial against diverse bacteria including multi-resistant Staphylococcus aureus (MRSA).

Pathogenic bacteria, such as multidrug-resistant Staphylococcus aureus (MRSA), which are not susceptible to most conventional antibiotics, are causing increased concern in healthcare institutions worldwide. The discovery of novel antibacterial compounds for biomedical exploitation is one avenue that is being pursued to combat these problematic bacteria. Marine eukaryotic microalgae are known to produce numerous useful products but have attracted little attention in the search for novel antibiotic compounds. Cell lysates of the marine diatom, Phaeodactylum tricornutum Bohlin, have been reported to display antibacterial activity in vitro, but the compounds responsible have not been fully identified. In this paper, using column chromatography and reversed-phase high-performance liquid chromatography, we report the isolation of an antibacterial fatty acid. Mass spectrometry and (1)H-nuclear magnetic resonance spectroscopy revealed it to be the polyunsaturated fatty acid, eicosapentaenoic acid (EPA). We show that EPA is active against a range of both Gram-positive and Gram-negative bacteria, including MRSA, at micromolar concentrations. These data indicate that it could find application in the topical and systemic treatment of drug-resistant bacterial infections.

Isolation and structural characterisation of two antibacterial free fatty acids from the marine diatom, Phaeodactylum tricornutum.

One solution to the global crisis of antibiotic resistance is the discovery of novel antimicrobial compounds for clinical application. Marine organisms are an attractive and, as yet, relatively untapped resource of new natural products. Cell extracts from the marine diatom, Phaeodactylum tricornutum, have antibacterial activity and the fatty acid, eicosapentaenoic acid (EPA), has been identified as one compound responsible for this activity. During the isolation of EPA, it became apparent that the extracts contained further antibacterial compounds. The present study was undertaken to isolate these additional antibacterial factors using silica column chromatography and reverse-phase high-performance liquid chromatography. Two antibacterial fractions, each containing a pure compound, were isolated and their chemical structures were investigated by mass spectrometry and nuclear magnetic resonance spectroscopy. The antibacterial compounds were identified as the monounsaturated fatty acid (9Z)-hexadecenoic acid (palmitoleic acid; C16:1 n-7) and the relatively unusual polyunsaturated fatty acid (6Z, 9Z, 12Z)-hexadecatrienoic acid (HTA; C16:3 n-4). Both are active against Gram-positive bacteria with HTA further inhibitory to the growth of the Gram-negative marine pathogen, Listonella anguillarum. Palmitoleic acid is active at micro-molar concentrations, kills bacteria rapidly, and is highly active against multidrug-resistant Staphylococcus aureus. These free fatty acids warrant further investigation as a new potential therapy for drug-resistant infections.

Crustin expression following bacterial injection and temperature change in the shore crab, Carcinus maenas.

The expression of carcinin, a crustin-type antimicrobial protein, in the crab, Carcinus maenas, was studied following in vivo challenge with Planococcus citreus, a Gram-positive bacterium known to be killed by the encoded protein. Real-time PCR analyses reveal that injection of P. citreus failed to elicit any significant changes in expression at 0-24h post-injection although there was a small, but significant, down-regulation at 84h in crabs held at 15 degrees C but not those at 5 or 20 degrees C. By contrast, un-injected crabs held at various temperatures between 5 and 20 degrees C, showed significantly up-regulated expression at 5 and at 20 but not 10 degrees C compared with controls at 15 degrees C. Thus expression of carcinin seems to be affected by temperature, especially when the animal is close to the edges of its physiologically tolerated thermal range.

Crustins: enigmatic WAP domain-containing antibacterial proteins from crustaceans.

Crustins are antibacterial proteins of ca. 7-14 kDa with a characteristic four-disulphide core-containing whey acidic protein (WAP) domain, expressed by the circulating haemocytes of crustaceans. Over 50 crustin sequences have been now reported from a variety of decapods, including crabs, lobsters, shrimp and crayfish. Three main types seem to occur but all possess a signal sequence at the amino terminus and a WAP domain at the carboxyl end. Differences between types lie in the structure of the central region. Those crustins purified as the native protein or expressed recombinantly all kill Gram-positive bacteria, and gene studies have shown that they are constitutively expressed, often at high levels, but show no consistent patterns of change in expression following injection of bacteria. This variable response to infection is enigmatic but indicates that these proteins could perform additional functions, perhaps as immune regulators in recovery from wounding, trauma or physiological stress.