Lulworthinone: In Vitro Mode of Action Investigation of an Antibacterial Dimeric Naphthopyrone Isolated from a Marine Fungus

Treatment options for infections caused by antimicrobial-resistant bacteria are rendered ineffective, and drug alternatives are needed—either from new chemical classes or drugs with new modes of action. Historically, natural products have been important contributors to drug discovery. In a recent study, the dimeric naphthopyrone lulworthinone produced by an obligate marine fungus in the family Lulworthiaceae was discovered. The observed potent antibacterial activity against Gram-positive bacteria, including several clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates, prompted this follow-up mode of action investigation. This paper aimed to characterize the antibacterial mode of action (MOA) of lulworthinone by combining in vitro assays, NMR experiments and microscopy. The results point to a MOA targeting the bacterial membrane, leading to improper cell division. Treatment with lulworthinone induced an upregulation of genes responding to cell envelope stress in Bacillus subtilis. Analysis of the membrane integrity and membrane potential indicated that lulworthinone targets the bacterial membrane without destroying it. This was supported by NMR experiments using artificial lipid bilayers. Fluorescence microscopy revealed that lulworthinone affects cell morphology and impedes the localization of the cell division protein FtsZ. Surface plasmon resonance and dynamic light scattering assays showed that this activity is linked with the compound‘s ability to form colloidal aggregates. Antibacterial agents acting at cell membranes are of special interest, as the development of bacterial resistance to such compounds is deemed more difficult to occur.     

Reactivation of broccoli peroxidases: structural changes of partially denatured isoenzymes

Structural changes involved in the reactivation of peroxidases (PODs) from broccoli and horseradish (HRP) following heat denaturation were investigated by using circular dichroism and absorption spectroscopy. Cooling heat-treated enzymes resulted in rapid refolding of the secondary structure into an inactive structural species, similar in conformation to the native enzyme. Reassociation of heme to the refolded peroxidase, as well as molecular rearrangement of the structure around the heme, occurs during incubation at approximately 25 degrees C and results in the return of biological activity. The secondary structure of neutral broccoli POD (N) is relatively heat labile, resulting in a rapid loss of activity, but the level of reactivation is high because the structure at the heme pocket is relatively stable. Acidic broccoli POD and HRP are more heat stable than N, but have a low degree of reactivation. Loss of activity is due primarily to alteration of the structure at the heme pocket. Effects of bovine serum albumin and pH on reactivation of PODs are also discussed. Keywords: Peroxidase; reactivation; horseradish; broccoli; circular dichroism; absorption spectroscopy.     

Smoke-derived taint in wine: effect of postharvest smoke exposure of grapes on the chemical composition and sensory characteristics of wine

Although smoke exposure has been associated with the development of smoke taint in grapes and subsequently in wine, to date there have been no studies that have demonstrated a direct link. In this study, postharvest smoke exposure of grapes was utilized to demonstrate that smoke significantly influences the chemical composition and sensory characteristics of wine and causes an apparent 'smoke taint'. Verdelho grapes were exposed to straw-derived smoke for 1 h and then fermented according to two different winemaking treatments. Control wines were made by fermenting unsmoked grapes. Sensory studies established a perceivable difference between smoked and unsmoked wines; smoked wines were described as exhibiting 'smoky', 'dirty', 'earthy', 'burnt' and 'smoked meat' characteristics. Quantitative analysis, by means of gas chromatography-mass spectrometry, identified guaiacol, 4-methylguaiacol, 4-ethylguaiacol, 4-ethylphenol, eugenol, and furfural in each of the wines made from smoked grapes. However, these compounds were not detected in the unsmoked wines, and their origin is therefore attributed to the application of smoke. Increased ethanol concentrations and browning were also observed in wines made from grapes exposed to smoke.     

Use of metabolic profiles in dairy cattle in tropical and subtropical countries on smallholder dairy farms

Metabolic profile testing has generally been used as part of a multidisciplinary approach for dairy herds in temperate climates. Our goal was to evaluate the effectiveness of the technique for identifying constraints on productivity in small herds in environments less favorable for milk production. Metabolites tested were chosen for stability in the sample after collection of blood, ease of analysis and practical knowledge of the meaning of the results. Blood levels of five different metabolites in low-producing dairy cows belonging to smallholders in tropical and subtropical environments were measured. The study involved 13 projects with 80 cows in each, carried out in six Latin American, six Asian, and one southern European countries. Data were also collected on feeding, body condition score (BCS) and weight change, parasitism, and reproduction. In Chile, Mexico, Paraguay, Philippines, Uruguay, and Venezuela, globulin levels were high in > 17% of cows sampled on each occasion. Globulin levels were also high in Turkey and Vietnam on one or more occasions. In Paraguay, 49% of cows had high globulin levels at two to three months after calving. These results suggest that inflammatory disease was present to a potentially important degree, although this was not always investigated and not always taken into account. In all countries except Mexico and Venezuela, high beta-hydroxybutyrate (BHB) levels before calving in many cows highlighted the presence of condition loss in late pregnancy, an important potential constraint on productivity and fertility. Fewer cows showed high BHB levels in lactation, whereas change in BCS and weight was more sensitive for measuring negative energy balance. Urea concentrations were low in only small numbers of cows suggesting that dietary protein shortages were not common. Albumin values were low mainly in cows where globulin values were high and, hence, did not generally provide additional information. The exception was in China where pregnant yaks over winter had high BHB and low albumin values, suggesting that they were seriously underfed. This observation stimulated a successful nutritional intervention in the following winter. Inorganic phosphate values were within the reference range in most countries a majority of the time suggesting, contrary to expectation, that this mineral was not commonly a constraint. The use of metabolic profile testing proved valuable in drawing attention to important potential constraints on productivity in dairy cows in tropical and subtropical environments and in confirming those which were not.     

Enzyme-linked immunosorbent assay (ELISA) and sol-gel-based immunoaffinity purification (IAP) of the pyrethroid bioallethrin in food and environmental samples

Enzyme-linked immunosorbent assay (ELISA) and sol-gel-based immunoaffinity purification (IAP) methods for the pyrethroid bioallethrin were developed and applied for monitoring bioallethrin in spiked food, soil, and dust samples. Attempts to determine bioallethrin content in fruit and vegetable extracts revealed high variability between sample preparations and marked interferences with the assay. Sol-gel IAP followed by solid-phase sample concentration was effective in removing the interfering components and resulted in high recovery of bioallethrin from spiked crude acetonic extracts of fruits and vegetables, even in the presence of high extract concentrations (28%). Solid-phase treatment alone failed to remove the interfering components from the spiked sample. Gas chromatography-mass spectrometry analysis of the IAP samples revealed bioallethrin as a doublet unsolved peak because of the cis and trans isomer present in the standard with confirmation of its mass. Unlike fruit and vegetable extracts, soil and dust samples did not interfere with the ELISA, and the bioallethrin content in those samples could be determined with high precision without the need of any further purification.     

Staphylococcus pseudintermedius in the dog: taxonomy, diagnostics, ecology, epidemiology and pathogenicity

The dog is the natural host of Staphylococcus pseudintermedius. Many research efforts are currently being undertaken to expand our knowledge and understanding of this important canine commensal and opportunistic pathogen. The objective of this review is to summarize the current knowledge of the species, including the latest research outcomes, with emphasis on taxonomy, diagnostics, ecology, epidemiology and pathogenicity. Despite the important taxonomic changes that have occurred over the past few years, the risk of misidentification in canine specimens is low and does not have serious consequences for clinical practice. Staphylococcus pseudintermedius carriage in the dog is more frequent and genetically heterogeneous compared with that of Staphylococcus aureus in man. It appears that these staphylococcal species have evolved separately through adaptation to their respective natural hosts and differ with regard to various aspects concerning ecology, population structure and evolution of antibiotic resistance. Further understanding of the ecology and epidemiology of S. pseudintermedius is hampered by the lack of a standard method for rapid and discriminatory typing and by the limited data available on longitudinal carriage and population structure of meticillin‐susceptible strains. With regard to pathogenicity, it is only now that we are starting to explore the virulence potential of S. pseudintermedius based on genomic and proteomic approaches, and more research is needed to assess the importance of individual virulence factors and the possible existence of hypervirulent strains.     

Virus-specific CD8⁺ T-cells detected in PBMC from horses vaccinated against African horse sickness virus

African horsesickness (AHS) is an infectious but noncontagious viral disease affecting all species of Equidae. The recall immune response of AHSV na?ve horses immunised with an attenuated African horsesickness virus serotype 4 (AHSV4) was characterised using immune assays including ELISPOT, real-time PCR (qPCR) and flow cytometry. The recall immune response detected in PBMC isolated from three inoculated horses showed an upregulation of circulating B lymphocytes that correlated with elevated IL-4 mRNA expression indicative of humoral immunity, but reduced frequency of CD4? cells. In addition to the expected antibody response, an increase in CD8? cells was also detected. Although these CD8? cells may be CTL, the role of these cells in immunity against AHSV still has to be determined.     

Microbial biogeography: from taxonomy to traits

The biogeographic variation of life has predominantly been studied using taxonomy, but this focus is changing. There is a resurging interest in understanding patterns in the distribution not only of taxa but also of the traits those taxa possess. Patterns of trait variation shed light on fundamental questions in biology, including why organisms live where they do and how they will respond to environmental change. Technological advances such as environmental genomics place microbial ecology in a unique position to move trait-based biogeography forward. We anticipate that as trait-based biogeography continues to evolve, micro- and macroorganisms will be studied in concert, establishing a science that is informed by and relevant to all domains of life.     

Evidence for positive epistasis in HIV-1

Reproductive strategies such as sexual reproduction and recombination that involve the shuffling of parental genomes for the production of offspring are ubiquitous in nature. However, their evolutionary benefit remains unclear. Many theories have identified potential benefits, but progress is hampered by the scarcity of relevant data. One class of theories is based on the assumption that mutations affecting fitness exhibit negative epistasis. Retroviruses recombine frequently and thus provide a unique opportunity to test these theories. Using amino acid sequence data and fitness values from 9466 human immunodeficiency virus 1 (HIV-1) isolates, we find in contrast to these theories strong statistical evidence for a predominance of positive epistasis in HIV-1.