The composition of the gut microbiota following early-life antibiotic exposure affects host health and longevity in later life.

Studies investigating whether there is a causative link between the gut microbiota and lifespan have largely been restricted to invertebrates or to mice with a reduced lifespan because of a genetic deficiency. We investigate the effect of early-life antibiotic exposure on otherwise healthy, normal chow-fed, wild-type mice, monitoring these mice for more than 700 days in comparison with untreated control mice. We demonstrate the emergence of two different low-diversity community types, post-antibiotic microbiota (PAM) I and PAM II, following antibiotic exposure. PAM II but not PAM I mice have impaired immunity, increased insulin resistance, and evidence of increased inflammaging in later life as well as a reduced lifespan. Our data suggest that differences in the composition of the gut microbiota following antibiotic exposure differentially affect host health and longevity in later life.

Biological Activities of Isolated Compounds from Three Edible Malaysian Red Seaweeds, Gracilaria changii, G. manilaensis and Gracilaria sp.

Gracilaria species are red marine macroalgae that are found abundantly in Malaysia. Gracilaria changii from Morib, Selangor, G. nanilaensis and Gracilaria sp. from Gelang Patah, Johor were used in this study. Five compounds were successfully isolated and identified as hexadecanoic acid (1), cholest-5-en-3-ol (2), 2-hydroxymyristic acid (3), cholesteryl myristate (4) and 1-(4'-methoxyphenyl)-3-(2",4",6"-trihydroxyphenyl)-3-hydroxypropanone (5) based on spectral data analysis (IR, UV, GC-MS, 'H NMR, "C NMR, HMQC and HMBC). All compounds isolated were tested for cytotoxicity (MTT assay for HL-60 and MCF-7 cell lines), and antibacterial (disc diffusion method), antioxidant (DPPH free radical scavenging assay and xanthine oxidase inhibitory assay) and acetylcholinesterase inhibitory (AChE) activity (TLC bioautographic method). Compounds I and 3 exhibited strong cytotoxic activity against HL-60 and MCF-7 cell lines. Compound 5 showed high antioxidant activity in both the DPPH free radical scavenging and xanthine oxidase inhibition assays. Compound I showed positive activity for AChE inhibitory with a minimum inhibition dose of 0.625 tg sample. All compounds demonstrated antibacterial activity producing 8 to 14 mm inhibition zones. A positive control was applied to all bioassays and experiments were performed with three replicates. Results demonstrated that three edible red seaweeds are rich sources of bioactive compounds with potential application for pharmaceutical purposes.

In-vitro neurotoxicity of two Malaysian krait species (Bungarus candidus and Bungarus fasciatus) venoms: neutralization by monovalent and polyvalent antivenoms from Thailand.

Bungarus candidus and Bungarus fasciatus are two species of krait found in Southeast Asia. Envenoming by these snakes is often characterized by neurotoxicity and, without treatment, causes considerable morbidity and mortality. In this study, the in vitro neurotoxicity of each species, and the effectiveness of two monovalent antivenoms and a polyvalent antivenom, against the neurotoxic effects of the venoms, were examined in a skeletal muscle preparation. Both venoms caused concentration-dependent inhibition of indirect twitches, and attenuated responses to exogenous nicotinic receptor agonists, in the chick biventer preparation, with B. candidus venom being more potent than B. fasciatus venom. SDS-PAGE and western blot analysis indicated different profiles between the venoms. Despite these differences, most proteins bands were recognized by all three antivenoms. Antivenom, added prior to the venoms, attenuated the neurotoxic effect of the venoms. Interestingly, the respective monovalent antivenoms did not neutralize the effects of the venom from the other Bungarus species indicating a relative absence of cross-neutralization. Addition of a high concentration of polyvalent antivenom, at the t90 time point after addition of venom, partially reversed the neurotoxicity of B. fasciatus venom but not B. candidus venom. The monovalent antivenoms had no significant effect when added at the t90 time point. This study showed that B. candidus and B. fasciatus venoms display marked in vitro neurotoxicity in the chick biventer preparation and administration of antivenoms at high dose is necessary to prevent or reverse neurotoxicity.

Proteomic characterization and comparison of Malaysian Bungarus candidus and Bungarus fasciatus venoms.

Kraits (Bungarus spp.) are highly venomous elapids that are only found in Asia. In the current study, 103 and 86 different proteins were identified from Bungarus candidus and Bungarus fasciatus venoms, respectively. These proteins were classified into 18 different venom protein families. Both venoms were found to contain a high percentage of three finger toxins, phospholipase A2 enzymes and Kunitz-type inhibitors. Smaller number of high molecular weight enzymes such as L-amino acid oxidase, hyaluronidases, and acetylcholinesterase were also detected in the venoms. We also detected some unique proteins that were not known to be present in these venoms. The presence of a natriuretic peptide, vespryn, and serine protease families was detected in B. candidus venom. We also detected the presence of subunit A and B of ß-bungarotoxin and α-bungarotoxin which had not been previously found in B. fasciatus venom. Understanding the proteome composition of Malaysian krait species will provide useful information on unique toxins and proteins which are present in the venoms. This knowledge will assist in the management of krait envenoming. In addition, these proteins may have potential use as research tools or as drug-design templates. BIOLOGICAL SIGNIFICANCE: This study has revealed the proteome composition of Malaysian B. candidus and B. fasciatus venoms, two medically important snake species in Asia. Information on the venom proteome of these species will provide useful information for krait bite management and aid in antivenom selection. Venom proteome profiles of these venoms showed that there are significant differences in the venom protein family compositions. Detection of proteins and peptides that have not been documented in these species such as natriuretic peptides, vespryn and serine proteases provides new knowledge on the composition of these venoms. The roles of these new proteins and peptides in krait envenoming are still unknown. Discovery of these proteins and peptides may also be useful for future research tool and therapeutic development.

Isolation and characterization of a presynaptic neurotoxin, P-elapitoxin-Bf1a from Malaysian Bungarus fasciatus venom.

Presynaptic neurotoxins are one of the major components in Bungarus venom. Unlike other Bungarus species that have been studied, ß-bungarotoxin has never been isolated from Bungarus fasciatus venom. It was hypothesized that the absence of ß-bungarotoxin in this species was due to divergence during evolution prior to evolution of ß-bungarotoxin. In this study, we have isolated a ß-bungarotoxin isoform we named P-elapitoxin-Bf1a by using gel filtration, cation-exchange and reverse-phase chromatography from Malaysian B. fasciatus venom. The toxin consists of two heterogeneous subunits, subunit A and subunit B. LCMS/MS data showed that subunit A was homologous to acidic phospholipase A2 subunit A3 from Bungarus candidus and B. multicinctus venoms, whereas subunit B was homologous with subunit B1 from B. fasciatus venom that was previously detected by cDNA cloning. The toxin showed concentration- and time-dependent reduction of indirect-twitches without affecting contractile responses to ACh, CCh or KCl at the end of experiment in the chick biventer preparation. Toxin modification with 4-BPB inhibited the neurotoxic effect suggesting the importance of His-48. Tissue pre-incubation with monovalent B. fasciatus (BFAV) or neuro-polyvalent antivenom (NPV), at the recommended titer, was unable to inhibit the twitch reduction induced by the toxin. This study indicates that Malaysian B. fasciatus venom has a unique ß-bungarotoxin isoform which was not neutralized by antivenoms. This suggests that there might be other presynaptic neurotoxins present in the venom and there is a variation in the enzymatic neurotoxin composition in venoms from different localities.