Rapid Detection of Botulinum Neurotoxins—A Review

A toxin is a poisonous substance produced within living cells or organisms. One of the most potent groups of toxins currently known are the Botulinum Neurotoxins (BoNTs). These are so deadly that as little as 62 ng could kill an average human; to put this into context that is approximately 200,000 × less than the weight of a grain of sand. The extreme toxicity of BoNTs leads to the need for methods of determining their concentration at very low levels of sensitivity. Currently the mouse bioassay is the most widely used detection method monitoring the activity of the toxin; however, this assay is not only lengthy, it also has both cost and ethical issues due to the use of live animals. This review focuses on detection methods both existing and emerging that remove the need for the use of animals and will look at three areas; speed of detection, sensitivity of detection and finally cost. The assays will have wide reaching interest, ranging from the pharmaceutical/clinical industry for production quality management or as a point of care sensor in suspected cases of botulism, the food industry as a quality control measure, to the military, detecting BoNT that has been potentially used as a bio warfare agent

Hierarchical Assembly of a Micro- and Macroporous Hydrogen-Bonded Organic Framework with Tailored Single-Crystal Size

Porous organic molecular materials represent an emergent field of research in Chemistry and Materials Science due to their unique combination of properties. To enhance their performance and expand the number of applications, the incorporation of hierarchical porosity is required, as exclusive microporosity entails several limitations. However, the integration of macropores in porous organic molecular materials is still an outstanding challenge. Herein, we report the first example of a hydrogen-bonded organic framework (MM-TPY) with hierarchical skeletal morphology, containing stable micro- and macroporosity. The crystal size, from micro to centimetre scale, can be controlled in a single step without using additives or templates. The mechanism of assembly during the crystal formation is compatible with a skeletal crystal growth. As proof of concept, we employed the hierarchical porosity as a platform for the dual, sequential and selective co-recognition of molecular species and microparticles.J.F.-S. thanks “Ramón y Cajal“ program (RYC2019-02794-I), MINECO (Spain) (Projects PID2019−104778GB−I00 and Excellence Unit “Maria de Maeztu” CEX2019−000919−M) and Generalitat Valenciana (SEJI/2020/034). E.V.R.F. thanks Ministerio de Ciencia e innovación (PID2020-116998RB-I00) and Ministerio de Educación y Formación Profesional (PRX21/00407)

Evaluating the use of 3'-(p-Aminophenyl) fluorescein for determining the formation of highly reactive oxygen species in particle suspensions

<p>Abstract</p> <p>Background</p> <p>Given the importance of highly reactive oxygen species (hROS) as reactants in a wide range of biological, photochemical, and environmental systems there is an interest in detection and quantification of these species. The extreme reactivity of the hROS, which includes hydroxyl radicals, presents an analytical challenge. 3'-(<it>p</it>-Aminophenyl) fluorescein (APF) is a relatively new probe used for measuring hROS. Here, we further evaluate the use of APF as a method for the detection of hydroxyl radicals in particle suspensions.</p> <p>Results</p> <p>Particle-generated hROS can be quantified with an estimated detection limit of 50 nM. Measurements of hROS in two National Institute of Standards and Technology (NIST 2709 and 2710) soil suspensions and a pyrite suspension show non-linear particle dose-response curves for hROS generation. APF can also be used in solutions containing no dissolved molecular oxygen (O₂) to determine the role of O₂in the formation of hROS. Results confirm that O₂is mechanistically important in the formation of hROS by dissolved ferrous iron and in pyrite suspensions.</p> <p>Conclusion</p> <p>Given the non-linear dose-response curves for particle generation of hROS, we recommend using several particle loadings in experiments aimed to compare particles for their hROS generation potential. The method presented here is specific to hROS and simple to perform. The analysis can be conducted in mobile labs as only basic laboratory equipment is required.</p

Rational Redesign of Glucose Oxidase for Improved Catalytic Function and Stability

Glucose oxidase (GOx) is an enzymatic workhorse used in the food and wine industries to combat microbial contamination, to produce wines with lowered alcohol content, as the recognition element in amperometric glucose sensors, and as an anodic catalyst in biofuel cells. It is naturally produced by several species of fungi, and genetic variants are known to differ considerably in both stability and activity. Two of the more widely studied glucose oxidases come from the species Aspergillus niger (A. niger) and Penicillium amagasakiense (P. amag.), which have both had their respective genes isolated and sequenced. GOx from A. niger is known to be more stable than GOx from P. amag., while GOx from P. amag. has a six-fold superior substrate affinity (KM) and nearly four-fold greater catalytic rate (kcat). Here we sought to combine genetic elements from these two varieties to produce an enzyme displaying both superior catalytic capacity and stability. A comparison of the genes from the two organisms revealed 17 residues that differ between their active sites and cofactor binding regions. Fifteen of these residues in a parental A. niger GOx were altered to either mirror the corresponding residues in P. amag. GOx, or mutated into all possible amino acids via saturation mutagenesis. Ultimately, four mutants were identified with significantly improved catalytic activity. A single point mutation from threonine to serine at amino acid 132 (mutant T132S, numbering includes leader peptide) led to a three-fold improvement in kcat at the expense of a 3% loss of substrate affinity (increase in apparent KM for glucose) resulting in a specify constant (kcat/KM) of 23.8 (mM−1 · s−1) compared to 8.39 for the parental (A. niger) GOx and 170 for the P. amag. GOx. Three other mutant enzymes were also identified that had improvements in overall catalysis: V42Y, and the double mutants T132S/T56V and T132S/V42Y, with specificity constants of 31.5, 32.2, and 31.8 mM−1 · s−1, respectively. The thermal stability of these mutants was also measured and showed moderate improvement over the parental strain

TLR9 expression in chronic lymphocytic leukemia identifies a promigratory subpopulation and novel therapeutic target

Chronic lymphocytic leukemia (CLL) remains incurable despite B-cell receptor–targeted inhibitors revolutionizing treatment. This suggests that other signaling molecules are involved in disease escape mechanisms and resistance. Toll-like receptor 9 (TLR9) is a promising candidate that is activated by unmethylated cytosine guanine dinucleotide–DNA. Here, we show that plasma from patients with CLL contains significantly more unmethylated DNA than plasma from healthy control subjects (P < .0001) and that cell-free DNA levels correlate with the prognostic markers CD38, β(2)-microglobulin, and lymphocyte doubling time. Furthermore, elevated cell-free DNA was associated with shorter time to first treatment (hazard ratio, 4.0; P = .003). We also show that TLR9 expression was associated with in vitro CLL cell migration (P < .001), and intracellular endosomal TLR9 strongly correlated with aberrant surface expression (sTLR9; r = 0.9). In addition, lymph node–derived CLL cells exhibited increased sTLR9 (P = .016), and RNA-sequencing of paired sTLR9(hi) and sTLR9(lo) CLL cells revealed differential transcription of genes involved in TLR signaling, adhesion, motility, and inflammation in sTLR9(hi) cells. Mechanistically, a TLR9 agonist, ODN2006, promoted CLL cell migration (P < .001) that was mediated by p65 NF-κB and STAT3 transcription factor activation. Importantly, autologous plasma induced the same effects, which were reversed by a TLR9 antagonist. Furthermore, high TLR9 expression promoted engraftment and rapid disease progression in a NOD/Shi-scid/IL-2Rγ(null) mouse xenograft model. Finally, we showed that dual targeting of TLR9 and Bruton’s tyrosine kinase (BTK) was strongly synergistic (median combination index, 0.2 at half maximal effective dose), which highlights the distinct role for TLR9 signaling in CLL and the potential for combined targeting of TLR9 and BTK as a more effective treatment strategy in this incurable disease

Inactivation of PNKP by mutant ATXN3 triggers apoptosis by activating the DNA damage-response pathway in SCA3.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an untreatable autosomal dominant neurodegenerative disease, and the most common such inherited ataxia worldwide. The mutation in SCA3 is the expansion of a polymorphic CAG tri-nucleotide repeat sequence in the C-terminal coding region of the ATXN3 gene at chromosomal locus 14q32.1. The mutant ATXN3 protein encoding expanded glutamine (polyQ) sequences interacts with multiple proteins in vivo, and is deposited as aggregates in the SCA3 brain. A large body of literature suggests that the loss of function of the native ATNX3-interacting proteins that are deposited in the polyQ aggregates contributes to cellular toxicity, systemic neurodegeneration and the pathogenic mechanism in SCA3. Nonetheless, a significant understanding of the disease etiology of SCA3, the molecular mechanism by which the polyQ expansions in the mutant ATXN3 induce neurodegeneration in SCA3 has remained elusive. In the present study, we show that the essential DNA strand break repair enzyme PNKP (polynucleotide kinase 3'-phosphatase) interacts with, and is inactivated by, the mutant ATXN3, resulting in inefficient DNA repair, persistent accumulation of DNA damage/strand breaks, and subsequent chronic activation of the DNA damage-response ataxia telangiectasia-mutated (ATM) signaling pathway in SCA3. We report that persistent accumulation of DNA damage/strand breaks and chronic activation of the serine/threonine kinase ATM and the downstream p53 and protein kinase C-d pro-apoptotic pathways trigger neuronal dysfunction and eventually neuronal death in SCA3. Either PNKP overexpression or pharmacological inhibition of ATM dramatically blocked mutant ATXN3-mediated cell death. Discovery of the mechanism by which mutant ATXN3 induces DNA damage and amplifies the pro-death signaling pathways provides a molecular basis for neurodegeneration due to PNKP inactivation in SCA3, and for the first time offers a possible approach to treatment.This study was funded by NIH grant NS073976 to TKH and a John Sealy Grant to PSS

TLR9 expression in Chronic Lymphocytic Leukemia identifies a pro-migratory subpopulation and novel therapeutic target

CLL remains incurable despite BCR-targeted inhibitors revolutionizing treatment. This suggests that other signaling molecules are involved in disease escape mechanisms and resistance. Toll-like receptor 9 (TLR9) is a promising candidate, which is activated by unmethylated CpG-DNA. Here, we show that plasma from CLL patients contains significantly more unmethylated DNA than plasma from healthy controls (p<0.0001) and that cell-free DNA levels correlate with the prognostic markers CD38, b2-microglobulin and lymphocyte doubling time. Furthermore, elevated cell-free DNA was associated with shorter time to first treatment (TTFT: p=0.003, HR=4.0). We went on to show that TLR9 expression was associated with in-vitro CLL cell migration (p<0.001) and intracellular endosomal TLR9 strongly correlated with aberrant surface expression ((sTLR9); r=0.9). In addition, lymph node-derived CLL cells showed increased sTLR9 (p=0.016) and RNA sequencing of paired sTLR9hi and sTLR9lo CLL cells revealed differential transcription of genes involved in TLR signaling, adhesion, motility and inflammation in sTLR9hi cells. Mechanistically, the TLR9 agonist, ODN2006, promoted CLL cell migration (p<0.001) that was mediated, by p65 NF-kB and STAT3 transcription factor activation. Importantly, autologous plasma induced the same effects, which were reversed by a TLR9 antagonist. Furthermore, high TLR9 expression promoted engraftment and rapid disease progression in a NSG mouse xenograft model. Finally, we showed that dual targeting of TLR9 and BTK was strongly synergistic (median CI=0.2 at ED50), which highlights the distinct role for TLR9 signaling in CLL and the potential for combined targeting of TLR9 and BTK as a more effective treatment strategy in this incurable disease