An antibody raised against a pathogenic serpin variant induces mutant-like behaviour in the wild-type protein.

A monoclonal antibody (mAb) that binds to a transient intermediate may act as a catalyst for the corresponding reaction; here we show this principle can extend on a macro-molecular scale to the induction of mutant-like oligomerisation in a wild-type protein. Using the common, pathogenic Glu342Lys (Z) variant of α1-antitrypsin as antigen - whose native state is susceptible to the formation of a proto-oligomeric intermediate - we have produced a mAb (5E3) that increases the rate of oligomerisation of the wild-type (M) variant. Employing ELISA, gel shift, thermal stability and FRET time-course experiments, we show that mAb5E3 does not bind to the native state of α1-antitrypsin, but recognises a cryptic epitope in the vicinity of the post-helix A loop and strand 4C that is revealed upon transition to the polymerisation intermediate, and which persists in the ensuing oligomer. This epitope is not shared by loop-inserted monomeric conformations. We show the increased amenity to polymerisation by either the pathogenic Glu342Lys mutation or the binding of mAb5E3 occurs without affecting energetic barrier to polymerisation. As mAb5E3 also does not alter the relative stability of the monomer to intermediate, it acts in a manner similar to the Glu342Lys mutation, by facilitating the conformational interchange between these two states

Development of a rapid, reliable and quantitative method: "SPOTi" for testing antifungal efficacy

A reference method for the antimicrobial susceptibility testing of common fungal pathogens such as dermatophytes, is currently lacking. In this study, we report the successful adaptation of solid agar-based spot culture growth inhibition assay (SPOTi) for dermatophytes, currently being used as a gold-standard in the anti-tubercular drug discovery field. The fungal-SPOTi assay correlated with the disc-diffusion method, and is validated using mycelial plugs. We propose the fungal-SPOTi as a high-throughput alternative to the disc-diffusion and broth micro-dilution anti-fungal assays to screen novel anti-fungals

An antibody raised against a pathogenic serpin variant induces mutant-like behaviour in the wild-type protein

A monoclonal antibody (mAb) that binds to a transient intermediate may act as a catalyst for the corresponding reaction; here we show this principle can extend on a macro molecular scale to the induction of mutant-like oligomerization in a wild-type protein. Using the common pathogenic E342K (Z) variant of α1-antitrypsin as antigen-whose native state is susceptible to the formation of a proto-oligomeric intermediate-we have produced a mAb (5E3) that increases the rate of oligomerization of the wild-type (M) variant. Employing ELISA, gel shift, thermal stability and FRET time-course experiments, we show that mAb5E3 does not bind to the native state of α1-antitrypsin, but recognizes a cryptic epitope in the vicinity of the post-helix A loop and strand 4C that is revealed upon transition to the polymerization intermediate, and which persists in the ensuing oligomer. This epitope is not shared by loop-inserted monomeric conformations. We show the increased amenity to polymerization by either the pathogenic E342K mutation or the binding of mAb5E3 occurs without affecting the energetic barrier to polymerization. As mAb5E3 also does not alter the relative stability of the monomer to intermediate, it acts in a manner similar to the E342K mutant, by facilitating the conformational interchange between these two states

Antagonism of Aspergillus terreus to Sclerotinia sclerotiorum.

An Aspergillus terreus strain showed in vitro antagonistic activity against tha plant pathogens Sclerotinia sclerotiorum (Lib.) de Bary.The interacton between A. terreus and sclerotia revealed that the mycoparasite sporulated abundantly on the sclerotial surface. Cell breakdown due to host cell wall disruption was observed in inner rind cells, by a scanning electron microscopy

Beyond RPE: The Perception of Exercise Under Normal and Ketotic Conditions

AimSubjective perceptions of exercising exertion are integral to maintaining homeostasis. Traditional methods have utilized scores of ‘rating of perceived exertion’ (RPE) to quantify these subjective perceptions, and here we aimed to test whether RPE may encompass identifiable localized perceptions from the lungs (breathlessness) and legs (leg discomfort), as well as their corresponding measures of anxiety. We utilized the intervention of ketoacidosis (via consumption of an exogenous ketone ester drink) to independently perturb exercise-related metabolites and humoral signals, thus allowing us to additionally identify the possible contributing physiological signals to each of these perceptions.MethodsTwelve trained volunteers underwent two incremental bicycle ergometer tests to exhaustion, following ingestion of either an exogenous ketone ester or a taste-matched placebo drink. Cardiorespiratory measures, blood samples and perceived exertion scales were taken throughout. Firstly, two-way repeated-measures ANOVAs were employed to identify the overall effects of ketoacidosis, followed by generalized linear mixed model regression to isolate physiological predictors contributing to each perception.ResultsRating of perceived exertion was found to contain contributions from localized perceptions of breathlessness and leg discomfort, with no measurable effect of ketoacidosis on overall exertion. Leg discomfort, anxiety of breathing and anxiety of leg discomfort were increased during ketoacidosis, and correspondingly contained pH within their prediction models. Anxiety of leg discomfort also encompassed additional humoral signals of blood glucose and ketone concentrations.ConclusionThese results indicate the presence of localized components of RPE in the form of breathlessness and leg discomfort. Furthermore, subjective perceptions of anxiety appear to result from a complex interplay of humoral signals, which may be evolutionarily important when monitoring exertion under times of metabolic stress, such as during starvation

Localization of Glycine Receptors in the Human Forebrain, Brainstem, and Cervical Spinal Cord: An Immunohistochemical Review

Inhibitory neurotransmitter receptors for glycine (GlyR) are heteropentameric chloride ion channels that are comprised of four functional subunits, alpha1–3 and beta and that facilitate fast-response, inhibitory neurotransmission in the mammalian brain and spinal cord. We have investigated the distribution of GlyRs in the human forebrain, brainstem, and cervical spinal cord using immunohistochemistry at light and confocal laser scanning microscopy levels. This review will summarize the present knowledge on the GlyR distribution in the human brain using our established immunohistochemical techniques. The results of our immunohistochemical labeling studies demonstrated GlyR immunoreactivity (IR) throughout the human basal ganglia, substantia nigra, various pontine regions, rostral medulla oblongata and the cervical spinal cord present an intense and abundant punctate IR along the membranes of the neuronal soma and dendrites. This work is part of a systematic study of inhibitory neurotransmitter receptor distribution in the human CNS, and provides a basis for additional detailed physiological and pharmacological studies on the inter-relationship of GlyR, GABAAR and gephyrin in the human brain. This basic mapping exercise, we believe, will provide important baselines for the testing of future pharmacotherapies and drug regimes that modulate neuroinhibitory systems. These findings provide new information for understanding the complexity of glycinergic functions in the human brain, which will translate into the contribution of inhibitory mechanisms in paroxysmal disorders and neurodegenerative diseases such as Epilepsy, Huntington's and Parkinson's Disease and Motor Neuron Disease