Synthetic substrate for application in both high and low throughput assays for botulinum neurotoxin B protease inhibitors

A FRET peptide substrate was synthesized and evaluated for enzymatic cleavage by the BoNT/B light chain protease. The FRET substrate was found to be useful in both a high throughput assay to uncover initial ‘hits’ and a low throughput HPLC assay to determine kinetic parameters and modes of inhibition.     

Gangliosides as High Affinity Receptors for Tetanus Neurotoxin

Tetanus neurotoxin (TeNT) is an exotoxin produced by Clostridium tetani that causes paralytic death to hundreds of thousands of humans annually. TeNT cleaves vesicle-associated membrane protein-2, which inhibits neurotransmitter release in the central nervous system to elicit spastic paralysis, but the molecular basis for TeNT entry into neurons remains unclear. TeNT is a ∼150-kDa protein that has AB structure-function properties; the A domain is a zinc metalloprotease, and the B domain encodes a translocation domain and C-terminal receptor-binding domain (HCR/T). Earlier studies showed that HCR/T bound gangliosides via two carbohydrate-binding sites, termed the lactose-binding site (the “W” pocket) and the sialic acid-binding site (the “R” pocket). Here we report that TeNT high affinity binding to neurons is mediated solely by gangliosides. Glycan array and solid phase binding analyses identified gangliosides that bound exclusively to either the W pocket or the R pocket of TeNT; GM1a bound to the W pocket, and GD3 bound to the R pocket. Using these gangliosides and mutated forms of HCR/T that lacked one or both carbohydrate-binding pocket, gangliosides binding to both of the W and R pockets were shown to be necessary for high affinity binding to neuronal and non-neuronal cells. The crystal structure of a ternary complex of HCR/T with sugar components of two gangliosides bound to the W and R supported the binding of gangliosides to both carbohydrate pockets. These data show that gangliosides are functional dual receptors for TeNT.Tetanus is an acute, often fatal disease of humans that was first described by Hippocrates over 24 centuries ago (1). Tetanus is characterized by generalized increased rigidity and convulsive spasms of skeletal muscles. Tetanus is caused by exposure to tetanus neurotoxin (TeNT)³ produced by the spore-forming bacterium Clostridium tetani. TeNT is delivered from the bloodstream to the peripheral nervous system, from where TeNT traffics to the central nervous system to cleave vesicle-associated membrane protein-2 (VAMP2), which inhibits neurotransmitter release and elicits spastic paralysis (2). Although prevented by vaccination, tetanus is responsible for hundreds of thousands of deaths per year in countries where vaccination is not common (3).TeNT is produced as a ∼150-kDa protein that is cleaved to a di-chain protein, comprising an N-terminal light chain (∼50 kDa) and a C-terminal heavy chain domain (∼100 kDa) linked through a single disulfide bond (4). TeNT light chain is a zinc metalloprotease that cleaves the neuronal SNARE protein VAMP2 (2). The TeNT heavy chain contains two functional domains: a translocation domain and a C-terminal receptor-binding domain (HCR/T, ∼50 kDa).The first step in TeNT action involves binding to a receptor(s) on the presynaptic membrane of α-motor neurons. Although the molecular basis for TeNT entry remains undetermined, an unambiguous role for gangliosides has been demonstrated (5–9). Current models implicate a dual receptor mechanism for the binding of the clostridial neurotoxins to neurons, which includes a ganglioside-binding component (10). Complex gangliosides are sialic acid-containing glycosphingolipids that are located on the outer leaflet of cell membranes and contain a common “core” (GA1) consisting of Gal(β1–3)GalNAc(β1–4)Gal(β1–4)Glc(β1–1)Cer to which one or more N-acetylneuraminic acids (sialic acids) are bound, yielding “a” and “b” series gangliosides (11, 12). Numerous structural and biochemical studies have established that HCR/T contains two carbohydrate-binding sites: a lactose-binding site and a sialic acid-binding site (13). Previous studies showed that Trp¹²⁸⁹ is the key residue for the lactose-binding site, and Arg¹²²⁶ is the key residue for the sialic acid-binding site (14). In this study, we denote the lactose-binding site as the “W” pocket and the sialic acid-binding site as the “R” pocket. Binz and co-workers (14) showed that functional R and W binding sites were required for TeNT toxicity (7). These biochemical and cellular studies were supported by a co-crystal structure of HCR/T bound to a GT1b-β anomer analog, which showed that the W and R carbohydrate-binding pockets were located at different regions of TeNT (7). We recently reported that the W pocket binds gangliosides via the GA1 core structure, whereas the R pocket binds gangliosides via di- or tri-sialic acid moieties (15) where simultaneous binding of TeNT to two gangliosides was synergistic (see Fig. 1a). In the current study, gangliosides were identified that bound exclusively to either the W pocket or R pocket, which allowed the characterization of the role of ganglioside binding to the W and R pockets as dual receptors for TeNT entry into neurons.Open in a separate windowFIGURE 1.Interaction of the HCR domain of TeNT with its putative cellular receptor. a, HCR/T has two ganglioside-binding sites. The W pocket binds to the terminal GalNAc-Gal of the ganglioside (illustrated by GM1a). The R pocket binds to the di-sialic acid of the ganglioside (illustrated by GD3). b, alternating lanes of molecular mass marker proteins and cortical neuron lysates were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was stained for protein with Ponceau S (bottom panel), and then the membrane strips were incubated with 10 nm of the indicated HCR/T (HCR/T wild type (wt), HCR/T (R+, W−), HCR/T (R−, W+), or HCR/T (R−, W−)) followed by HRP-conjugated α-FLAG antibody. The bands were visualized with SuperSignal; exposed film is shown (upper panel). The asterisk denotes the position of purified gangliosides resolved under identical conditions. Migration of the molecular mass marker proteins is indicated (kDa) in the left-most lane in the upper panel.     

A Short History of Biosemiotics

Biosemiotics is the synthesis of biology and semiotics, and its main purpose is to show that semiosis is a fundamental component of life, i.e., that signs and meaning exist in all living systems. This idea started circulating in the 1960s and was proposed independently from enquires taking place at both ends of the Scala Naturae. At the molecular end it was expressed by Howard Pattee’s analysis of the genetic code, whereas at the human end it took the form of Thomas Sebeok’s investigation into the biological roots of culture. Other proposals appeared in the years that followed and gave origin to different theoretical frameworks, or different schools, of biosemiotics. They are: (1) the physical biosemiotics of Howard Pattee and its extension in Darwinian biosemiotics by Howard Pattee and by Terrence Deacon, (2) the zoosemiotics proposed by Thomas Sebeok and its extension in sign biosemiotics developed by Thomas Sebeok and by Jesper Hoffmeyer, (3) the code biosemiotics of Marcello Barbieri and (4) the hermeneutic biosemiotics of Anton Marko?. The differences that exist between the schools are a consequence of their different models of semiosis, but that is only the tip of the iceberg. In reality they go much deeper and concern the very nature of the new discipline. Is biosemiotics only a new way of looking at the known facts of biology or does it predict new facts? Does biosemiotics consist of testable hypotheses? Does it add anything to the history of life and to our understanding of evolution? These are the major issues of the young discipline, and the purpose of the present paper is to illustrate them by describing the origin and the historical development of its main schools.     

The Background of Mitochondrial DNA Haplogroup J Increases the Sensitivity of Leber's Hereditary Optic Neuropathy Cells to 2,5-Hexanedione Toxicity

Leber''s hereditary optic neuropathy (LHON) is a maternally inherited blinding disease due to mitochondrial DNA (mtDNA) point mutations in complex I subunit genes, whose incomplete penetrance has been attributed to both genetic and environmental factors. Indeed, the mtDNA background defined as haplogroup J is known to increase the penetrance of the 11778/ND4 and 14484/ND6 mutations. Recently it was also documented that the professional exposure to n-hexane might act as an exogenous trigger for LHON. Therefore, we here investigate the effect of the n-hexane neurotoxic metabolite 2,5-hexanedione (2,5-HD) on cell viability and mitochondrial function of different cell models (cybrids and fibroblasts) carrying the LHON mutations on different mtDNA haplogroups. The viability of control and LHON cybrids and fibroblasts, whose mtDNAs were completely sequenced, was assessed using the MTT assay. Mitochondrial ATP synthesis rate driven by complex I substrates was determined with the luciferine/luciferase method. Incubation with 2,5-HD caused the maximal loss of viability in control and LHON cells. The toxic effect of this compound was similar in control cells irrespective of the mtDNA background. On the contrary, sensitivity to 2,5-HD induced cell death was greatly increased in LHON cells carrying the 11778/ND4 or the 14484/ND6 mutation on haplogroup J, whereas the 11778/ND4 mutation in association with haplogroups U and H significantly improved cell survival. The 11778/ND4 mutation on haplogroup U was also more resistant to inhibition of complex I dependent ATP synthesis by 2,5-HD. In conclusion, this study shows that mtDNA haplogroups modulate the response of LHON cells to 2,5-HD. In particular, haplogroup J makes cells more sensitive to its toxic effect. This is the first evidence that an mtDNA background plays a role by interacting with an environmental factor and that 2,5-HD may be a risk element for visual loss in LHON. This proof of principle has broad implications for other neurodegenerative disorders such as Parkinson''s disease.     

Carbon accumulation in agricultural soils after afforestation: a meta-analysis

Deforestation usually results in significant losses of soil organic carbon (SOC). The rate and factors determining the recovery of this C pool with afforestation are still poorly understood. This paper provides a review of the influence of afforestation on SOC stocks based on a meta-analysis of 33 recent publications (totaling 120 sites and 189 observations), with the aim of determining the factors responsible for the restoration of SOC following afforestation. Based on a mixed linear model, the meta-analysis indicates that the main factors that contribute to restoring SOC stocks after afforestation are: previous land use, tree species planted, soil clay content, preplanting disturbance and, to a lesser extent, climatic zone. Specifically, this meta-analysis (1) indicates that the positive impact of afforestation on SOC stocks is more pronounced in cropland soils than in pastures or natural grasslands; (2) suggests that broadleaf tree species have a greater capacity to accumulate SOC than coniferous species; (3) underscores that afforestation using pine species does not result in a net loss of the whole soil-profile carbon stocks compared with initial values (agricultural soil) when the surface organic layer is included in the accounting; (4) demonstrates that clay-rich soils (> 33%) have a greater capacity to accumulate SOC than soils with a lower clay content (< 33%); (5) indicates that minimizing preplanting disturbances may increase the rate at which SOC stocks are replenished; and (6) suggests that afforestation carried out in the boreal climate zone results in small SOC losses compared with other climate zones, probably because trees grow more slowly under these conditions, although this does not rule out gains over time after the conversion. This study also highlights the importance of the methodological approach used when developing the sampling design, especially the inclusion of the organic layer in the accounting.     

Fermentative production of self-toxic fungal secondary metabolites

Fungi are well known for their vast diversity of secondary metabolites that include many life-saving drugs and highly toxic mycotoxins. In general, fungal cultures producing such metabolites are immune to their toxic effects. However, some are known to produce self-toxic compounds that can pose production optimization challenges if the metabolites are needed in large amounts for chemical modification. One such culture, LV-2841, was identified as the lead for one of our exploratory projects. This culture was found to be a slow grower that produced trace amounts of a known metabolite, cercosporamide, under the standard flask fermentation conditions, and extensive medium optimization studies failed to yield higher titers. Poor growth of the culture in liquid media was attributed to the self-toxicity of cercosporamide to the producing organism, and the minimum inhibitory concentration (MIC) of cercosporamide was estimated to be in the range of 8–16 μg/ml. Fermentations carried out in media containing Diaion^® HP20 resin afforded significantly higher titers of the desired compound. While several examples of resin-based fermentations of soil streptomyces have been published, this approach has rarely been used for fungal fermentations. Over a 100-fold increase in the production titer of cercosporamide, a self-toxic secondary metabolite, was achieved by supplementing the production medium with a commercially available neutral adsorbent resin.     

A forward genetic screen identifies mutants deficient for mitochondrial complex I assembly in Chlamydomonas reinhardtii

Mitochondrial complex I is the largest multimeric enzyme of the respiratory chain. The lack of a model system with facile genetics has limited the molecular dissection of complex I assembly. Using Chlamydomonas reinhardtii as an experimental system to screen for complex I defects, we isolated, via forward genetics, amc1-7 nuclear mutants (for assembly of mitochondrial complex I) displaying reduced or no complex I activity. Blue native (BN)-PAGE and immunoblot analyses revealed that amc3 and amc4 accumulate reduced levels of the complex I holoenzyme (950 kDa) while all other amc mutants fail to accumulate a mature complex. In amc1, -2, -5-7, the detection of a 700 kDa subcomplex retaining NADH dehydrogenase activity indicates an arrest in the assembly process. Genetic analyses established that amc5 and amc7 are alleles of the same locus while amc1-4 and amc6 define distinct complementation groups. The locus defined by the amc5 and amc7 alleles corresponds to the NUOB10 gene, encoding PDSW, a subunit of the membrane arm of complex I. This is the first report of a forward genetic screen yielding the isolation of complex I mutants. This work illustrates the potential of using Chlamydomonas as a genetically tractable organism to decipher complex I manufacture.     

Thermoregulation of alpacas bred in Italy

The present study monitored daily and seasonal variations of rectal temperature in response to different environmental temperatures in alpacas bred in the Italian Apennines at 300 m a.s.l. In each season, the rectal temperature of 33 clinically healthy alpacas was measured three times/day (morning, midday, afternoon). Ambient temperatures were also recorded. Rectal temperatures ranged from a minimum value of 35.1 to a maximum of 39.4°C, with a maximum daily thermal excursion (ΔT_rec) of 3.2°C. Temperatures increased throughout the day, with highly significant differences recorded in both young and adult animals between all the time bands (P < 0.001). These differences were particularly dramatic for adults in summer, when the mean rectal temperature in the morning was 36.3 ± 0.13°C, probably as a consequence of recent shearing. Significant ΔT_rec differences were recorded depending on the season in both young and adult animals (P < 0.001), with the highest ΔT_rec values recorded in summer (although the highest daily ambient excursion value was recorded in winter). In conclusion, similarly to alpacas bred in their natural environment, alpacas bred in Italy show a wide thermal neutrality zone, which is probably an adaptive response, that allows the animals to save energy. In the Italian Apennines, in order to prevent situations of hypothermia, with possible detrimental effects on alpacas’ health and welfare, shearing should be carried out only in warm seasons.