Structural Insights into Rational Design of Single-Domain Antibody-Based Antitoxins against Botulinum Neurotoxins.

Botulinum neurotoxin (BoNT) is one of the most acutely lethal toxins known to humans, and effective treatment for BoNT intoxication is urgently needed. Single-domain antibodies (VHH) have been examined as a countermeasure for BoNT because of their high stability and ease of production. Here, we investigate the structures and the neutralization mechanisms for six unique VHHs targeting BoNT/A1 or BoNT/B1. These studies reveal diverse neutralizing mechanisms by which VHHs prevent host receptor binding or block transmembrane delivery of the BoNT protease domain. Guided by this knowledge, we design heterodimeric VHHs by connecting two neutralizing VHHs via a flexible spacer so they can bind simultaneously to the toxin. These bifunctional VHHs display much greater potency in a mouse co-intoxication model than similar heterodimers unable to bind simultaneously. Taken together, our studies offer insight into antibody neutralization of BoNTs and advance our ability to design multivalent anti-pathogen VHHs with improved therapeutic properties

Neuromuscular recovery from botulism involves multiple forms of compensatory plasticity

IntroductionBotulinum neurotoxin (BoNT) causes neuroparalytic disease and death by blocking neuromuscular transmission. There are no specific therapies for clinical botulism and the only treatment option is supportive care until neuromuscular function spontaneously recovers, which can take weeks or months after exposure. The highly specialized neuromuscular junction (NMJ) between phrenic motor neurons and diaphragm muscle fibers is the main clinical target of BoNT. Due to the difficulty in eliciting respiratory paralysis without a high mortality rate, few studies have characterized the neurophysiological mechanisms involved in diaphragm recovery from intoxication. Here, we develop a mouse model of botulism that involves partial paralysis of respiratory muscles with low mortality rates, allowing for longitudinal analysis of recovery.Methods and resultsMice challenged by systemic administration of 0.7 LD50 BoNT/A developed physiological signs of botulism, such as respiratory depression and reduced voluntary running activity, that persisted for an average of 8–12 d. Studies in isolated hemidiaphragm preparations from intoxicated mice revealed profound reductions in nerve-elicited, tetanic and twitch muscle contraction strengths that recovered to baseline 21 d after intoxication. Despite apparent functional recovery, neurophysiological parameters remained depressed for 28 d, including end plate potential (EPP) amplitude, EPP success rate, quantal content (QC), and miniature EPP (mEPP) frequency. However, QC recovered more quickly than mEPP frequency, which could explain the discrepancy between muscle function studies and neurophysiological recordings. Hypothesizing that differential modulation of voltage-gated calcium channels (VGCC) contributed to the uncoupling of QC from mEPP frequency, pharmacological inhibition studies were used to study the contributions of different VGCCs to neurophysiological function. We found that N-type VGCC and P/Q-type VGCC partially restored QC but not mEPP frequency during recovery from paralysis, potentially explaining the accelerated recovery of evoked release versus spontaneous release. We identified additional changes that presumably compensate for reduced acetylcholine release during recovery, including increased depolarization of muscle fiber resting membrane potential and increased quantal size.DiscussionIn addition to identifying multiple forms of compensatory plasticity that occur in response to reduced NMJ function, it is expected that insights into the molecular mechanisms involved in recovery from neuromuscular paralysis will support new host-targeted treatments for multiple neuromuscular diseases

Statistical Study of Mercury’s Energetic Electron Events as Observed by the Gamma‐Ray and Neutron Spectrometer Instrument Onboard MESSENGER

We present results from a statistical analysis of Mercury’s energetic electron (EE) events as observed by the gamma‐ray and neutron spectrometer instrument onboard the MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) spacecraft. The main objective of this study is to investigate possible anisotropic behavior of EE events using multiple data sets from MESSENGER instruments. We study the data from the neutron spectrometer (NS) and the gamma‐ray spectrometer anticoincidence shield (ACS) because they use the same type of borated plastic scintillator and, hence, they have very similar response functions, and their large surface areas make them more sensitive to low‐intensity EE events than MESSENGER’s particle instrumentation. The combined analysis of NS and ACS data reveals two different classes of energetic electrons: “Standard” events and “ACS‐enhanced” events. Standard events, which comprise over 90% of all events, have signal sizes that are the same in both the ACS and NS. They are likely gyrating particles about Mercury’s magnetic field following a 90° pitch angle distribution and are located in well‐defined latitude and altitude regions within Mercury’s magnetosphere. ACS‐enhanced events, which comprise less than 10% of all events, have signal sizes in the ACS that are 10 to 100 times larger than those observed by the NS. They follow a beam‐like distribution and are observed both inside and outside Mercury’s magnetosphere with a wider range of latitudes and altitudes than Standard events. The difference between the Standard and ACS‐enhanced event characteristics suggests distinct underyling acceleration mechanisms.Key PointsA comprehensive survey of energetic electron (EE) events observed with the neutron spectrometer (NS) and the gamma‐ray spectrometer anticoincidence shield (ACS) is conductedThe majority of EE events detected in the NS are also detected in the ACS and appear to be composed of gyrating, drifting electronsACS‐only and ACS‐enhanced events exhibit a significantly different spatial and temporal characteristics compared with the other EE event classesPeer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/145319/1/jgra54299_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/145319/2/jgra54299.pd

Comprehensive survey of energetic electron events in Mercury\u27s magnetosphere with data from the MESSENGER Gamma-Ray and Neutron Spectrometer

Data from the MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) Gamma-Ray and Neutron Spectrometer have been used to detect and characterize energetic electron (EE) events in Mercury\u27s magnetosphere. This instrument detects EE events indirectly via bremsstrahlung photons that are emitted when instrument and spacecraft materials stop electrons having energies of tens to hundreds of keV. From Neutron Spectrometer data taken between 18 March 2011 and 31 December 2013 we have identified 2711 EE events. EE event amplitudes versus energy are distributed as a power law and have a dynamic range of a factor of 400. The duration of the EE events ranges from tens of seconds to nearly 20 min. EE events may be classified as bursty (large variation with time over an event) or smooth (small variation). Almost all EE events are detected inside Mercury\u27s magnetosphere on closed field lines. The precise occurrence times of EE events are stochastic, but the events are located in well-defined regions with clear boundaries that persist in time and form what we call “quasi-permanent structures.” Bursty events occur closer to dawn and at higher latitudes than smooth events, which are seen near noon-to-dusk local times at lower latitudes. A subset of EE events shows strong periodicities that range from hundreds of seconds to tens of milliseconds. The few-minute periodicities are consistent with the Dungey cycle timescale for the magnetosphere and the occurrence of substorm events in Mercury\u27s magnetotail region. Shorter periods may be related to phenomena such as north-south bounce processes for the energetic electrons

First observations of Mercury's plasma mantle by MESSENGER

We present the first observations of Mercury's plasma mantle, a primary region for solar wind entry into the planetary magnetosphere, located in the high‐latitude magnetotail. MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) observations from two orbits on 10 November 2012 have been analyzed. The main plasma mantle features are (1) a steady decrease in proton density as MESSENGER moved deeper into the magnetotail; (2) frequent flux transfer events throughout the magnetosheath and into the magnetotail, suggesting that these events are the primary source for solar wind plasma injection; (3) a diamagnetic depression, due to the presence of plasma, as pressure balance is maintained; and (4) a clear proton velocity dispersion, resulting from lower‐energy protons being transported deep into the magnetosphere as higher‐energy protons escape downtail. From these velocity dispersions we infer cross‐magnetosphere electric potentials of 23 kV and 29 kV, consistent with estimates determined from measurements of magnetopause reconnection rate and tail loading and unloading events

Comprehensive survey of energetic electron events in Mercury's magnetosphere with data from the MESSENGER Gamma‐Ray and Neutron Spectrometer

Data from the MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) Gamma‐Ray and Neutron Spectrometer have been used to detect and characterize energetic electron (EE) events in Mercury's magnetosphere. This instrument detects EE events indirectly via bremsstrahlung photons that are emitted when instrument and spacecraft materials stop electrons having energies of tens to hundreds of keV. From Neutron Spectrometer data taken between 18 March 2011 and 31 December 2013 we have identified 2711 EE events. EE event amplitudes versus energy are distributed as a power law and have a dynamic range of a factor of 400. The duration of the EE events ranges from tens of seconds to nearly 20 min. EE events may be classified as bursty (large variation with time over an event) or smooth (small variation). Almost all EE events are detected inside Mercury's magnetosphere on closed field lines. The precise occurrence times of EE events are stochastic, but the events are located in well‐defined regions with clear boundaries that persist in time and form what we call “quasi‐permanent structures.” Bursty events occur closer to dawn and at higher latitudes than smooth events, which are seen near noon‐to‐dusk local times at lower latitudes. A subset of EE events shows strong periodicities that range from hundreds of seconds to tens of milliseconds. The few‐minute periodicities are consistent with the Dungey cycle timescale for the magnetosphere and the occurrence of substorm events in Mercury's magnetotail region. Shorter periods may be related to phenomena such as north‐south bounce processes for the energetic electrons

Neuronal delivery of antibodies has therapeutic effects in animal models of botulism = La administración de anticuerpos en neuronas tiene efectos terapéuticos en modelos animales del botulismo

Abstract: Botulism is caused by a potent neurotoxin that blocks neuromuscular transmission, resulting in death by asphyxiation. Currently, the therapeutic options are limited and there is no antidote. Here, we harness the structural and trafficking properties of an atoxic derivative of botulinum neurotoxin (BoNT) to transport a function-blocking single-domain antibody into the neuronal cytosol where it can inhibit BoNT serotype A (BoNT/A1) molecular toxicity. Post-symptomatic treatment relieved toxic signs of botulism and rescued mice, guinea pigs, and nonhuman primates after lethal BoNT/A1 challenge. This platform might enable delivery of antibodies and other protein-based therapeutics to previously inaccessible intraneuronal targets. Resumen: El botulismo es causado por una potente neurotoxina que bloquea la transmisión neuromuscular, provocando la muerte por asfixiamiento. Actualmente, las opciones de tratamiento son limitadas y no existe un antídoto. En este artículo aprovechamos las propiedades estructurales y de tráfico neuronal de un derivado atóxico de la neurotoxina botulínica (BoNT) para transportar un anticuerpo que bloquea la función al citosol neuronal, donde puede inhibir la toxicidad molecular del serotipo A de la BoNT (BoNT /A1). El tratamiento post-sintomático alivió los signos tóxicos del botulismo y rescató de la muerte a ratones, conejillos de indias y primates no humanos después de la exposición letal a BoNT / A1. Esta plataforma podría permitir la administración de anticuerpos y otros terapéuticos basados en proteínas a blancos intraneuronales que previamente eran inaccesibles