Botulinum Neurotoxin Induces Neurotoxic Microglia Mediated by Exogenous Inflammatory Responses.

Botulinum neurotoxin serotype A (BoNT/A) is widely used in therapeutics and cosmetics. The effects of multi-dosed BoNT/A treatment are well documented on the peripheral nervous system (PNS), but much less is known on the central nervous system (CNS). Here, the mechanism of multi-dosed BoNT/A leading to CNS neurodegeneration is explored by using the 3D human neuron-glia model. BoNT/A treatment reduces acetylcholine, triggers astrocytic transforming growth factor beta, and upregulates C1q, C3, and C5 expression, inducing microglial proinflammation. The disintegration of the neuronal microtubules is escorted by microglial nitric oxide, interleukin 1ß, tumor necrosis factor α, and interleukin 8. The microglial proinflammation eventually causes synaptic impairment, phosphorylated tau (pTau) aggregation, and the loss of the BoNT/A-treated neurons. Taking a more holistic approach, the model will allow to assess therapeutics for the CNS neurodegeneration under the prolonged use of BoNT/A.

Critical analysis in the advancement of cell-based assays for botulinum neurotoxin.

The study on botulinum neurotoxins (BoNTs) has rapidly evolved for their structure and functions as opposed to them being poisons or cures. Since their discoveries, the scientific community has come a long way in understanding BoNTs' structure and biological activity. Given its current application as a tool for understanding neurocellular activity and as a drug against over 800 neurological disorders, relevant and sensitive assays have become critical for biochemical, physiological, and pharmacological studies. The natural entry of the toxin being ingestion, it has also become important to examine its mechanism while crossing the epithelial cell barrier. Several techniques and methodologies have been developed, for its entry, pharmacokinetics, and biological activity for identification, and drug efficacy both in vivo and in vitro conditions. However, each of them presents its own challenges. The cell-based assay is a platform that exceeds the sensitivity of mouse bioassay while encompassing all the steps of intoxication including cell binding, transcytosis, endocytosis, translocation and proteolytic activity. In this article we review in detail both the neuronal and nonneuronal based cellular interaction of BoNT involving its transportation, and interaction with the targeted cells, and intracellular activities.

Land use and land cover dynamics and traditional agroforestry practices in Wonchi District, Ethiopia.

BACKGROUND: Investigating the land use and land cover (LULC) dynamics and the status of traditional agroforestry practices provide important data for policymakers. The main objective of this study was to assess the LULC dynamics and traditional agroforestry practices among smallholder farmers across the two agro-ecological zones in Wonchi District of Ethiopia. METHODS: Landsat images were acquired from Earth Explorer, and changes in LULC were quantified with three Landsat sensors in the three time-series (1985, 2001, and 2019). Supervised classification with maximum likelihood technique was employed using ERDAS Imagine and ArcGIS. A ground survey was conducted with 100 key informants who were selected from 10 sites using a purposive sampling method. The collected data were subjected to direct matrix ranking, use-value analysis of most important multipurpose plant species, and semi-structured interviews were conducted for qualitative analysis. RESULTS: In total, 103 agroforestry plant species belonging to 44 families were identified in Wonchi District, of which 74 were indigenous including seven endemic and 29 exotic species. The highest species (13) were recorded in the Fabaceae family. About 61% of species were reported in the midland agro-ecological zone. A mixed farming system was the most frequently (56%) reported source of income. The results of LULC changes from 1985 to 2019 showed that the agroforestry cover increased from 31.1% to 34.9% and settlement including road construction increased from 12.5% to 31.6% of the total area with an annual rate change of 0.3% and 2.7%, respectively. These changes corresponded with a decreasing trend of the forest, cropland, water body, and shrub at a rate of 4.7%, 1.3%, 0.8%, and 0.5%, respectively. The LULC changes were more pronounced in the highlands than in the midlands of Wonchi District. Expansion of settlement and tenure policy change are the main drivers for these changes in the area. The authors recommended that protecting and planting indigenous and multipurpose plant species is essential as restoration techniques for all degraded land-use types. Therefore, strengthening agroforestry practices and land-use planning is urgently needed for achieving multiple goals.

Clostridial Neurotoxins: Structure, Function and Implications to Other Bacterial Toxins.

Gram-positive bacteria are ancient organisms. Many bacteria, including Gram-positive bacteria, produce toxins to manipulate the host, leading to various diseases. While the targets of Gram-positive bacterial toxins are diverse, many of those toxins use a similar mechanism to invade host cells and exert their functions. Clostridial neurotoxins produced by Clostridial tetani and Clostridial botulinum provide a classical example to illustrate the structure-function relationship of bacterial toxins. Here, we critically review the recent progress of the structure-function relationship of clostridial neurotoxins, including the diversity of the clostridial neurotoxins, the mode of actions, and the flexible structures required for the activation of toxins. The mechanism clostridial neurotoxins use for triggering their activity is shared with many other Gram-positive bacterial toxins, especially molten globule-type structures. This review also summarizes the implications of the molten globule-type flexible structures to other Gram-positive bacterial toxins. Understanding these highly dynamic flexible structures in solution and their role in the function of bacterial toxins not only fills in the missing link of the high-resolution structures from X-ray crystallography but also provides vital information for better designing antidotes against those toxins.

Carbon stocks of above- and belowground tree biomass in Kibate Forest around Wonchi Crater Lake, Central Highland of Ethiopia.

Forests play an important role in the global carbon (C) balance, but their biomass has decreased globally mainly because of deforestation and a reduction in forest cover. However, little is known about the C stock of tree biomass related to environmental factors in the remnant forest patches. Thus, the present study aimed at assessing the status of C stocks of tree biomass using an allometric equation in Kibate Forest (Ethiopia). Sixty-six plots (30×30 m) were laid out at 100 m interval distance along the altitudinal gradients in five transects. The results revealed that the highest C stocks (67.4%) per species were contributed by Juniperus procera, Ilex mitis var. mitis, Nuxia congesta, and Olea europaea subsp. cuspidata. The mean total tree biomass was 91.9 ± 10.01 Mg ha-1. The mean total C stock was 45.9 ± 5.17 Mg ha-1, out of which 38.3 ± 4.31 and 7.7 ± 0.91 Mg ha-1 were stored in above- and belowground C pools, respectively. Anthropogenic factors were negatively associated with the C-stock distribution in the study area. Thus, the status of the C stock of tree biomass related to anthropogenic factors indicates that sustainable forest management practice is needed in the study area to conserve biodiversity and mitigate climate change.

Botulinum neurotoxin inhibitor binding dynamics and kinetics relevant for drug design.

BACKGROUND: A natural product analog, 3-(4-nitrophenyl)-7H-furo[3,2-g]chromen-7-one, which is a nitrophenyl psoralen (NPP) was found to be an effective inhibitor of botulinum neurotoxin type A (BoNT/A). METHODS: In this work, we performed enzyme inhibition kinetics and employed biochemical techniques such as isothermal calorimetry (ITC) and fluorescence spectroscopy as well as molecular modeling to examine the kinetics and binding mechanism of NPP inhibitor with BoNT/A LC. RESULTS: Studies of inhibition mechanism and binding dynamics of NPP to BoNT/A light chain (BoNT/A LC) showed that NPP is a mixed type inhibitor for the zinc endopeptidase activity, implying that at least part of the inhibitor-enzyme binding site may be different from the substrate-enzyme binding site. By using biochemical techniques, we demonstrated NPP forms a stable complex with BoNT/A LC. These observations were confirmed by Molecular Dynamics (MD) simulation, which demonstrates that NPP binds to the site near the active site. CONCLUSION: The NPP binding interferes with BoNT/A LC binding to the SNAP-25, hence, inhibits its cleavage. Based on these results, we propose a modified strategy for designing a molecule to enhance the efficiency of the inhibition against the neurotoxic effect of BoNT. GENERAL SIGNIFICANCE: Insights into the interactions of NPP with BoNT/A LC using biochemical and computational approaches will aid in the future development of effective countermeasures and better pharmacological strategies against botulism.

Biofortification of Silage Maize with Zinc, Iron and Selenium as Affected by Nitrogen Fertilization.

Agronomic biofortification is one of the main strategies for alleviation of micronutrient deficiencies in human populations and promoting sustainable production of food and feed. The aim of this study was to investigate the effect of nitrogen (N)fertilization on biofortification of maize crop (Zea mays L.) with zinc (Zn), iron (Fe) and selenium (Se) grown on a micronutrient deficient soil under greenhouse conditions. Factorial design experiment was set under greenhouse conditions. The experiment consisted of two levels of each N, Zn, Fe and Se. The levels for N were 125 and 250 mg N kg-1 soil; Zn were 1 and 5 mg Zn kg-1 soil; levels of Fe were 0 and 10 mg Fe kg-1 soil; levels of Se were 0 and 0.02 mg Se kg-1 soil. An additional experiment was also conducted to study the effect of the Zn form applied as a ZnO or ZnSO4 on shoot growth, shoot Zn concentration and total shoot Zn uptake per plant. Shoot Zn concentrations increased by increasing soil Zn application both with ZnSO4 and ZnO treatments, but the shoot Zn concentration and total Zn uptake were much greater with ZnSO4 than the ZnO application. Under given experimental conditions, increasing soil N supply improved shoot N concentration; but had little effect on shoot dry matter production. The concentrations of Zn and Fe in shoots were significantly increased by increasing N application. In case of total uptake of Zn and Fe, the positive effect of N nutrition was more pronounced. Although Se soil treatment had significant effect, N application showed no effect on Se concentration and accumulation in maize shoots. The obtained results show that N fertilization is an effective tool in improving the Zn and Fe status of silage maize and contribute to the better-quality feed.

Botulinum Endopeptidase: SAXS Experiments and MD Simulations Reveal Extended Solution Structures That Account for Its Biochemical Properties.

Development of antidotes against botulism requires understanding of the enzymatically active conformations of Botulinum neurotoxin serotype A (BoNT/A) light chain (LCA). We performed small angle X-ray scattering (SAXS) to characterize the solution structures of truncated light chain (tLCA). The 34-37 Å radius of gyration of tLCA was 1.5-times greater than the averaged 22-23-Å radius from the crystal structures. The bimodal distribution of interatomic distances P(r) indicated the two-domain tLCA structure with 129-133 Å size, and Kratky plots indicated the tLCA partial unfolding in the 25-37 °C temperature range. To interpret these data, we employed molecular dynamics simulations and machine learning. Excellent agreement between experimental and theoretical P(r) profiles helped to resolve conformational subpopulations of tLCA in solution. Partial unfolding of the C-terminal portion of tLCA (residues 339-425) results in formation of extended conformations with the larger globular domain (residues 2-298) and the smaller unstructured domain (339-425). The catalytic domain, buried 20 Å-deep inside the crystal structure, becomes accessible in extended solution conformations (8-9 Å deep). The C- and N-termini containing different functional sequence motifs are maximally separated in the extended conformations. Our results offer physical insights into the molecular basis of BoNT/A function and stress the importance of reversible unfolding-refolding transitions and hydrophobic interactions.

A Phenotypic Switch of Differentiated Glial Cells to Dedifferentiated Cells Is Regulated by Folate Receptor α.

In a previous study, we showed that folate receptor-α (FRα) translocates to the nucleus where it acts as a transcription factor and upregulates Hes1, Oct4, Sox2, and Klf4 genes responsible for pluripotency. Here, we show that acetylation and phosphorylation of FRα favor its nuclear translocation in the presence of folate and can cause a phenotypic switch from differentiated glial cells to dedifferentiated cells. shRNA-FRα mediated knockdown of FRα was used to confirm the role of FRα in dedifferentiation. Ocimum sanctum hydrophilic fraction-1 treatment not only blocks the folate mediated dedifferentiation of glial cells but also promotes redifferentiation of dedifferentiated glial cells, possibly by reducing the nuclear translocation of ~38 kDa FRα and subsequent interaction with chromatin assembly factor-1. Stem Cells 2019;37:1441-1454.

Post - Mining soil as carbon storehouse under polish conditions.

The main aim of these studies was to determine the potential for carbon sequestration in brown coal open-cast mine by phytoremediation using scots pine (Pinus sylvestris L.) and giant miscanthus (Miscanthus x giganteus) plants. This paper presents relationships between soil organic carbon (SOC) sequestration and carbon phytosequestration in waste dump associated with open-cast lignite mine in Central Poland. The research is the continuation of previously carried out experiments, but was conducted in field conditions. In reclamation of post-mining landscapes, during field experiment, an effect of sewage sludge, compost and lake chalk amendments and in combination of plants was investigated. The impact of soil amendments on carbon stock, CO2 emission reduction, plant biomass production and carbon content in shoots and roots was studied. The highest SOC stock was found in soil treated with sewage sludge (33 Mg*ha-1) and compost (45 Mg*ha-1) stabilized by lake chalk. These fertilizer combinations also contributed the most in relation to CO2 emission reduction through SOC stock (83 Mg*ha-1 and 127 Mg*ha-1 respectively). In addition, greater amounts (60-100%) of soil organic matter was converted into humic acids fraction. This phenomenon could be the initial stage of the progressive process of organic matter deposition and carbon sequestration in post-mining area. Carbon phytosequestration was determined through carbon bound in plant tissues. The highest carbon content (60%) in both plant species was recorded in treatments with sewage sludge and compost with lake chalk. Stabilization of compost by lake chalk application was good method to improve the efficiency of carbon sequestration in soil and carbon phytosequestration. Improving the efficiency of these two processes, through skillfully selected soil additives and plant species, may be used on a larger scale in the future as an alternative to the storage of carbon dioxide, especially in degraded areas.

Evolutionary Features in the Structure and Function of Bacterial Toxins.

Toxins can function both as a harmful and therapeutic molecule, depending on their concentrations. The diversity in their function allows us to ask some very pertinent questions related to their origin and roles: (a) What makes them such effective molecules? (b) Are there evolutionary features encoded within the structures of the toxins for their function? (c) Is structural hierarchy in the toxins important for maintaining their structure and function? (d) Do protein dynamics play a role in the function of toxins? and (e) Do the evolutionary connections to these unique features and functions provide the fundamental points in driving evolution? In light of the growing evidence in structural biology, it would be appropriate to suggest that protein dynamics and flexibility play a much bigger role in the function of the toxin than the structure itself. Discovery of IDPs (intrinsically disorder proteins), multifunctionality, and the concept of native aggregation are shaking the paradigm of the requirement of a fixed three-dimensional structure for the protein's function. Growing evidence supporting the above concepts allow us to redesign the structure-function aspects of the protein molecules. An evolutionary model is necessary and needs to be developed to study these important aspects. The criteria for a well-defined model would be: (a) diversity in structure and function, (b) unique functionality, and (c) must belong to a family to define the evolutionary relationships. All these characteristics are largely fulfilled by bacterial toxins. Bacterial toxins are diverse and widely distributed in all three forms of life (Bacteria, Archaea and Eukaryotes). Some of the unique characteristics include structural folding, sequence and functional combination of domains, targeting a cellular process to execute their function, and most importantly their flexibility and dynamics. In this work, we summarize certain unique aspects of bacterial toxins, including role of structure in defining toxin function, uniqueness in their enzymatic function, and interaction with their substrates and other proteins. Finally, we have discussed the evolutionary aspects of toxins in detail, which will help us rethink the current evolutionary theories. A careful study, and appropriate interpretations, will provide answers to several questions related to the structure-function relationship of proteins, in general. Additionally, this will also allow us to refine the current evolution theories.

Natural Compounds and Their Analogues as Potent Antidotes against the Most Poisonous Bacterial Toxin.

Botulinum neurotoxins (BoNTs), the most poisonous proteins known to humankind, are a family of seven (serotype A to G) immunologically distinct proteins synthesized primarily by different strains of the anaerobic bacterium Clostridium botulinum Being the causative agents of botulism, the toxins block neurotransmitter release by specifically cleaving one of the three soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, thereby inducing flaccid paralysis. The development of countermeasures and therapeutics against BoNTs is a high-priority research area for public health because of their extreme toxicity and potential for use as biowarfare agents. Extensive research has focused on designing antagonists that block the catalytic activity of BoNTs. In this study, we screened 300 small natural compounds and their analogues extracted from Indian plants for their activity against BoNT serotype A (BoNT/A) as well as its light chain (LCA) using biochemical and cellular assays. One natural compound, a nitrophenyl psoralen (NPP), was identified to be a specific inhibitor of LCA with an in vitro 50% inhibitory concentration (IC50) value of 4.74 ± 0.03 µM. NPP was able to rescue endogenous synaptosome-associated protein 25 (SNAP-25) from cleavage by BoNT/A in human neuroblastoma cells with an IC50 of 12.2 ± 1.7 µM, as well as to prolong the time to the blocking of neutrally elicited twitch tensions in isolated mouse phrenic nerve-hemidiaphragm preparations.IMPORTANCE The long-lasting endopeptidase activity of BoNT is a critical biological activity inside the nerve cell, as it prompts proteolysis of the SNARE proteins, involved in the exocytosis of the neurotransmitter acetylcholine. Thus, the BoNT endopeptidase activity is an appropriate clinical target for designing new small-molecule antidotes against BoNT with the potential to reverse the paralysis syndrome of botulism. In principle, small-molecule inhibitors (SMIs) can gain entry into BoNT-intoxicated cells if they have a suitable octanol-water partition coefficient (log P) value and other favorable characteristics (P. Leeson, Nature 481:455-456, 2012, https://doi.org/10.1038/481455a). Several efforts have been made in the past to develop SMIs, but inhibitors effective under in vitro conditions have not in general been effective in vivo or in cellular models (L. M. Eubanks, M. S. Hixon, W. Jin, S. Hong, et al., Proc Natl Acad Sci U S A 104:2602-2607, 2007, https://doi.org/10.1073/pnas.0611213104). The difference between the in vitro and cellular efficacy presumably results from difficulties experienced by the compounds in crossing the cell membrane, in conjunction with poor bioavailability and high cytotoxicity. The screened nitrophenyl psoralen (NPP) effectively antagonized BoNT/A in both in vitro and ex vivo assays. Importantly, NPP inhibited the BoNT/A light chain but not other general zinc endopeptidases, such as thermolysin, suggesting high selectivity for its target. Small-molecule (nonpeptidic) inhibitors have better oral bioavailability, better stability, and better tissue and cell permeation than antitoxins or peptide inhibitors.

Woody species composition and diversity of riparian vegetation along the Walga River, Southwestern Ethiopia.

The primary objective of this study was to examine the status of woody species composition and diversity along the Walga River of Wonchi, Southwestern Ethiopia. Fifty quadrats of 10 m x 50 m were laid at 500 m interval through systematic sampling method along the river line. Vegetation height (≥2.5 m) and DBH (≥2.5 cm) of only tree species were measured and altitude, ecological disturbances such as, grazing intensity and human impacts were included as main environmental variables at each of the sampled plots. The data was analyzed using different R statistical packages. Ninety-nine woody vascular plant species belonged to 81 genera and 45 familieswere recorded in Walga riparian vegetation. Only 10% of specieswere endemic to the Flora area. Asteraceae and Fabaceae had the highest number of species. Majority of the species (52.5%) were shrubs. Four major plant community types were identified: Euclea divinorum-Maytenus arbutifolia (1), Pterolobium stellatum- Calpurnia aurea (2), Brucea antidysenterica-Prunus africana (3), Erica arborea-Hagenia abyssinica (4). Species richness, true diversity and importance values were highestin community type 2(the lowest altitude ranges between 1976-2212 m a.s.l.) while evenness was highestin community type 3(mid altitude ranges between 2359-2676 m a.s.l.). Both community typeswere comprised of 56% of all recorded species and all endemic taxa except two. The highest percentage of species in lower frequency classes indicates a higher degree of floristic heterogeneity. There was a strong negative correlation (r = -0.65, p<0.001) between species richness and altitude with 42% of the variation in species richness per plot being explained by altitude. Our findings suggest that human disturbances and excessive livestock grazing are the main threats in community types1 and 2. We conclude that identifying major plant community types and underlying environmental conditions may help to manage and conserve forest resources in the area.

Role of critical elements in botulinum neurotoxin complex in toxin routing across intestinal and bronchial barriers.

The highly potent botulinum neurotoxin serotype A (BoNT/A) inhibits neurotransmitter release at neuromuscular junctions resulting in flaccid muscle paralysis, respiratory arrest and death. In order to reach their neuronal cell targets, BoNT/A must cross epithelial cell barriers lining the intestines and airways. The toxin is produced as a large protein complex comprised of the neurotoxin and non-toxic neurotoxin-associated proteins (NAPs). Although NAPs are known to protect the toxin from harsh environments, their role in the movement of BoNT/A across epithelial barriers has not been fully characterized. In the current study, movement of the toxin across epithelial cells was examined macroscopically using a sensitive near infrared fluorescence transcytosis assay and microscopically using fluorescently labeled toxin and confocal microscopy. The studies show that the BoNT/A complex internalizes more rapidly than the pure toxin. The studies also show that one NAP protein, hemaglutinin 33 (Hn33), enhanced both the binding and movement of a deactivated recombinant botulinum neurotoxin A (DrBoNT) across epithelial cell monolayers and that the toxin associates with Hn33 on the cell surface. Collectively, the data demonstrate that, in addition to their protective role, NAPs and Hn33 play an important role in BoNT/A intoxication.

A novel role of C-terminus in introducing a functionally flexible structure critical for the biological activity of botulinum neurotoxin.

Botulinum neurotoxin (BoNT) is responsible for botulism, a clinical condition resulting in flaccid muscle paralysis and potentially death. The light chain is responsible for its intracellular toxicity through its endopeptidase activity. Available crystal structures of BoNT/A light chains (LCA) are based on various truncated versions (tLCA) of the full-length LCA (fLCA) and do not necessarily reflect the true structure of LCA in solution. The understanding of the mechanism of action, longevity of intoxication, and an improved development of endopeptidase inhibitors are dependent on first having a better insight into the structure of LCA in solution. Using an array of biophysical techniques, we report that the fLCA structure is significantly more flexible than tLCA in solution, which may be responsible for its dramatically higher enzymatic activity. This seems to be achieved by a much stronger, more rapid binding to substrate (SNAP-25) of the fLCA compared to tLCA. These results suggest that the C-terminus of LCA plays a critical role in introducing a flexible structure, which is essential for its biological function. This is the first report of such a massive structural role of the C-terminus of a protein being critical for maintaining a functional state.

Effect of nitrogen and zinc fertilization on zinc and iron bioavailability and chemical speciation in maize silage.

Agronomic biofortification is one of the main strategies for alleviation of micronutrient deficiencies in food and feed. The objective of this study was to investigate the effect of N supply on total concentration of Zn and Fe and their chemical species in the soluble extracts of maize silage grown under field conditions. Total concentrations of Zn, Fe, Cu, Mn, S and P were measured by flow-injection inductive coupled plasma (ICP) - mass spectrometer (MS). Soluble Fe and Zn were extracted and analyzed by size exclusion-inductively coupled plasma mass spectrometry. Using the same set-up for total elemental and speciation analysis enabled direct quantitative comparison of the detected speciated molecules with the total element sample content. N or Zn treatment, except in control plots, did not significantly affect concentrations of Zn and Fe in the maize silage and grain samples. Significant positive correlation was observed between Zn and Fe maize silage (r = 0.64, p < 0.01) and maize grain (r = 0.85, p < 0.01) concentrations. N and Zn treatment did not affect solubility of Zn and Fe, while available Zn and Fe were affected by increase in Zn soil treatment. Soluble Zn was speciated in LMW complexes, while soluble Fe was speciated in MMW and LMW complexes.

Differential endopeptidase activity of different forms of type A botulinum neurotoxin: A unique relationship between the size of the substrate and activity of the enzyme.

Botulinum neurotoxins (BoNTs; serotypes A-G) are metalloproteases, which cleave and inactivate cellular proteins essential for neurotransmitter release. In bacterial cultures, BoNTs are secreted as a complex of the neurotoxin and a group of neurotoxin associated proteins (NAPs). Under physiological condition (pH 7.4), this complex is believed to be dissociated to separate the neurotoxin from NAPs. BoNT consists of a 50 kDa light (L) chain (LC or catalytic domain) and a 100 kDa heavy (H) chain (or HC) linked through a disulfide bond and other non-covalent interactions. The cell intoxication involves three major steps; binding, membrane translocation and inhibition of neurotransmitter release. The last step of intoxication, endopeptidase activity, is very unique and specific that can be used for detection of the complex and isolated forms of the toxin. A fluorescent tag-labeled synthetic peptide (SNAPtide) derived from a segment of SNAP-25, an intracellular substrate of BoNT/A, is used to detect and assay the endopeptidase activity of BoNT/A. The detection of the signal is based on the change in the fluorescence energy transfer after selective cleavage of the peptide by the BoNT/A. In this report, we demonstrate that SNAPtide as a commonly used substrate widely differ in reaction with BoNT/A complex, BoNT/A, and BoNT/A light chain. These findings have implications for assays used in detection, and in screening potential inhibitors.

High Yield Preparation of Functionally Active Catalytic-Translocation Domain Module of Botulinum Neurotoxin Type A That Exhibits Uniquely Different Enzyme Kinetics.

Botulinum neurotoxins (BoNTs) are the most toxic proteins known to cause flaccid muscle paralysis as a result of inhibition of neurotransmitter release from peripheral cholinergic synapses. BoNT type A (BoNT/A) is a 150 kDa protein consisting of two major subunits: light chain (LC) and heavy chain (HC). The LC is required for the catalytic activity of neurotoxin, whereas the C and N terminal domains of the HC are required for cell binding, and translocation of LC across the endosome membranes, respectively. To better understand the structural and functional aspects of BoNT/A intoxication we report here the development of high yield Escherichia coli expression system (2-20-fold higher yield than the value reported in the literature) for the production of recombinant light chain-translocation domain (rLC-TD/A) module of BoNT/A which is catalytically active and translocation competent. The open reading frame of rLC-TD/A was PCR amplified from deactivated recombinant BoNT/A gene (a non-select agent reagent), and was cloned using pET45b (+) vector to express in E. coli cells. The purification procedure included a sequential order of affinity chromatography, trypsinization, and anion exchange column chromatography. We were able to purify > 95% pure, catalytically active and structurally well-folded protein. Comparison of enzyme kinetics of purified LC-TD/A to full-length toxin and recombinant light chain A suggest that the affinity for the substrate is in between endopeptidase domain and botulinum toxin. The potential application of the purified protein has been discussed in toxicity and translocation assays.

A Novel Surface Plasmon Resonance Biosensor for the Rapid Detection of Botulinum Neurotoxins.

Botulinum neurotoxins (BoNTs) are Category A agents on the NIAID (National Institute of Allergy and Infectious Diseases) priority pathogen list owing to their extreme toxicity and the relative ease of production. These deadly toxins, in minute quantities (estimated human i.v. lethal dose LD50 of 1-2 ng/kg body weight), cause fatal flaccid paralysis by blocking neurotransmitter release. The current gold standard detection method, the mouse-bioassay, often takes days to confirm botulism. Furthermore, there are no effective antidotes known to reverse the symptoms of botulism, and as a result, patients with severe botulism often require meticulous care during the prolonged paralytic illness. To combat potential bio-terrorism incidents of botulinum neurotoxins, their rapid detection is paramount. Surface plasmon resonance (SPR) is a very sensitive technique to examine bio-molecular interactions. The label-free, real-time analysis, with high sensitivity and low sample consumption makes this technology particularly suitable for detection of the toxin. In this study, we demonstrated the feasibility in an assay with a newly designed SPR instrument for the rapid detection of botulinum neurotoxins. The LOD (limit of detection) of the Newton Photonics (NP) SPR based assay is 6.76 pg/mL for Botulinum Neurotoxin type A Light Chain (BoNT/A LC). We established that the detection sensitivity of the system is comparable to the traditional mouse LD50 bioassay in BoNT/A using this SPR technology.

Sewage sludge disposal strategies for sustainable development.

The main objective of the present review is to compare the existing sewage sludge management solutions in terms of their environmental sustainability. The most commonly used strategies, that include treatment and disposal has been favored within the present state-of-art, considering existing legislation (at European and national level), characterization, ecotoxicology, waste management and actual routs used currently in particular European countries. Selected decision making tools, namely End-of-waste criteria and Life Cycle Assessment has been proposed in order to appropriately assess the possible environmental, economic and technical evaluation of different systems. Therefore, some basic criteria for the best suitable option selection has been described, in the circular economy "from waste to resources" sense. The importance of sewage sludge as a valuable source of matter and energy has been appreciated, as well as a potential risk related to the application of those strategies.