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The GluN2C subunit exists predominantly, but not exclusively in NMDA receptors within the cerebellum. Antagonists such as UBP1700 and positive allosteric modulators including PYD-106 and 3-acylamino-2-aminopropionic acid derivatives such as UA3-10 (( R )-2-amino-3-{[5-(2-bromophenyl)thiophen-2-yl]carboxamido}propionic acid) represent promising tool compounds to investigate the role of GluN2C-containing NMDA receptors in the signal transduction in the brain. However, due to its high polarity the bioavailability and CNS penetration of the amino acid UA3-10 are expected to be rather low. Herein, three ester prodrugs 12a - c of the NMDA receptor glycine site agonist UA3-10 were prepared and pharmacokinetically characterized. The esters 12a - c showed higher lipophilicity (higher log D 7.4 values) than the acid UA3-10 but almost the same binding at human serum albumin. The acid UA3-10 was rather stable upon incubation with mouse liver microsomes and NADPH, but the esters 12a - c were fast hydrolyzed to afford the acid UA3-10. Incubation with pig liver esterase and mouse serum led to rapid hydrolysis of the esters 12a - c . The isopropyl ester 12c showed a promising log D 7.4 value of 3.57 and the highest stability in the presence of pig liver esterase and mouse serum. These results demonstrate that ester prodrugs of UA3-10 can potentially afford improved bioavailability and CNS penetration., (© 2022 Walter de Gruyter GmbH, Berlin/Boston.)

Botulism is a paralytic disease caused by botulinum neurotoxins (BoNTs). Equine antitoxin is currently the standard therapy for botulism in human. The preparation of equine antitoxin relies on the immunization of horses with botulinum toxoid, which suffers from low yield and safety limitations. The Hc fragment of BoNTs was suggested to be a potent antibotulinum subunit vaccine. The current study presents a comparative evaluation of equine-based toxoid-derived antitoxin (TDA) and subunit-derived antitoxin (SDA). The potency of recombinant Hc/A, Hc/B, and Hc/E in mice was similar to that of toxoids of the corresponding serotypes. A single boost with Hc/E administered to a toxoid E-hyperimmune horse increased the neutralizing antibody concentration (NAC) from 250 to 850 IU/mL. Immunization of naïve horses with the recombinant subunits induced a NAC comparable to that of horses immunized with the toxoid. SDA and TDA bound common epitopes on BoNTs, as demonstrated by an in vitro competition binding assay. In vivo, SDA and TDA showed similar efficacy when administered to guinea pigs postexposure to a lethal dose of botulinum toxins. Collectively, the results of the current study suggest that recombinant BoNT subunits may replace botulinum toxoids as efficient and safe antigens for the preparation of pharmaceutical anti-botulinum equine antitoxins.

Purpose: The purpose of this study is to compare perioperative and oncological outcomes of upfront vs. delayed early radical cystectomy (eRC) for high-risk non-muscle-invasive bladder cancer (HR-NMIBC). Methods: All consecutive HR-NMIBC patients who underwent eRC between 2001 and 2020 were retrospectively included and divided into upfront and delayed groups, according to the receipt or not of BCG. Perioperative outcomes were evaluated and the impact of upfront vs. delayed eRC on pathological upstaging, defined as ≥pT2N0 disease at final pathology, was assessed using multivariable logistic regression. Recurrence-free (RFS), cancer-specific (CSS) and overall survival (OS) were compared between upfront and delayed eRC groups using inverse probability of treatment weighting (IPTW)-adjusted Cox model. Results: Overall, 184 patients received either upfront (n = 87; 47%) or delayed (n = 97; 53%) eRC. No difference was observed in perioperative outcomes between the two treatment groups (all p > 0.05). Pathological upstaging occurred in 55 (30%) patients and upfront eRC was an independent predictor (HR = 2.65; 95% CI = (1.23−5.67); p = 0.012). In the IPTW-adjusted Cox analysis, there was no significant difference between upfront and delayed eRC in terms of RFS (HR = 1.31; 95% CI = (0.72−2.39); p = 0.38), CSS (HR = 1.09; 95% CI = (0.51−2.34); p = 0.82) and OS (HR = 1.19; 95% CI = (0.62−2.78); p = 0.60). Conclusion: our results suggest similar perioperative outcomes between upfront and delayed eRC, with an increased risk of upstaging after upfront eRC that did impact survival, as compared to delayed eRC.

The receptor-binding domain of botulinum neurotoxin (H C fragment), is a promising botulism vaccine candidate. In the current study, fermentation strategies were evaluated to upscale H C fragment expression. A simple translation of the growth conditions from shake flasks to a batch fermentation process resulted in limited culture growth and protein expression (OD of 11 and volumetric protein yields of 123 mg/L). Conducting fed-batch fermentation with rich media and continuous nutrient supplementation significantly improved culture growth (OD of 40.3) and protein expression (1093 mg/L). A further increase in H C fragment yield was achieved by high cell density cultivation (HCDC). The bacterium was grown in a defined medium and with a combined bolus/continuous feed of nutrients to maintain desired oxygen levels and prevent acetate accumulation. The final OD of the process was 260, and the volumetric yield of the H C fragment was 2065 mg/L, which reflects improvement by an order of magnitude. Purified H C fragments, produced by HCDC, exhibited typical biochemical and protective characteristics in mice. Taken together, the advancements achieved in this study promote large-scale production of the H C fragment in E. coli for use in anti-botulism vaccines.

Botulinum neurotoxin type E (BoNT/E), the fastest acting toxin of all BoNTs, cleaves the 25 kDa synaptosomal-associated protein (SNAP-25) in motor neurons, leading to flaccid paralysis. The specific detection and quantification of the BoNT/E-cleaved SNAP-25 neoepitope can facilitate the development of cell-based assays for the characterization of anti-BoNT/E antibody preparations. In order to isolate highly specific monoclonal antibodies suitable for the in vitro immuno-detection of the exposed neoepitope, mice and rabbits were immunized with an eight amino acid peptide composed of the C-terminus of the cleaved SNAP-25. The immunized rabbits developed a specific and robust polyclonal antibody response, whereas the immunized mice mostly demonstrated a weak antibody response that could not discriminate between the two forms of SNAP-25. An immune scFv phage-display library was constructed from the immunized rabbits and a panel of antibodies was isolated. The sequence alignment of the isolated clones revealed high similarity between both heavy and light chains with exceptionally short HCDR3 sequences. A chimeric scFv-Fc antibody was further expressed and characterized, exhibiting a selective, ultra-high affinity (pM) towards the SNAP-25 neoepitope. Moreover, this antibody enabled the sensitive detection of cleaved SNAP-25 in BoNT/E treated SiMa cells with no cross reactivity with the intact SNAP-25. Thus, by applying an immunization and selection procedure, we have isolated a novel, specific and high-affinity antibody against the BoNT/E-derived SNAP-25 neoepitope. This novel antibody can be applied in in vitro assays that determine the potency of antitoxin preparations and reduce the use of laboratory animals for these purposes.

The pharmacopeia mouse neutralization assay (PMNA) is the standard method for determining the potency of phar­maceutical botulinum antitoxins. However, a PMNA requires a large number of mice, and, thus, an alternative in vitro method to replace it is needed. Herein, we developed an in vitro SiMa cell line-based neutralization assay (SBNA), compatible with a PMNA design, for therapeutic antitoxins against type E botulinum neurotoxin (BoNT/E). The SBNA measures the residual cellular activity of BoNT/E following antitoxin neutralization in the SiMa lysate using a specific quantitative sandwich ELISA for its cleaved cellular target protein SNAP-25. The potencies of different pharmaceutical antitoxin preparations were determined by applying two different quantification approaches: (1) a cutoff value, in accor­dance with the pharmacopeia concept, and (2) nonlinear regression of a standard curve generated by serial dilutions of a standard antitoxin. Both approaches achieved accurate potencies compared to the PMNA (average %RE of ~16%). Furthermore, the SBNA was able to determine in vitro, for the first time, the accurate neutralizing activity (%RE ≤ 20) of next-generation equine and rabbit therapeutic antitoxins. Collectively, a high correlation between SBNA and PMNA results was obtained for all antitoxin preparations (r = 0.99, P < 0.0001 for the standard curve approach, and r = 0.97, p < 0.0001 for the cutoff approach). In conclusion, the SBNA can potentially replace the PMNA and markedly reduce the need for laboratory animals for the approval of botulinum antitoxin preparations.

Antitoxin, the only licensed drug therapy for botulism, neutralizes circulating botulinum neurotoxin (BoNT). However, antitoxin is no longer effective when a critical amount of BoNT has already entered its target nerve cells. The outcome is a chronic phase of botulism that is characterized by prolonged paralysis. In this stage, blocking toxin activity within cells by next-generation intraneuronal anti-botulinum drugs (INABDs) may shorten the chronic phase of the disease and accelerate recovery. However, there is a lack of adequate animal models that simulate the chronic phase of botulism for evaluating the efficacy of INABDs. Herein, we report the development of a rabbit model for the chronic phase of botulism, induced by intoxication with a sublethal dose of BoNT. Spirometry monitoring enabled us to detect deviations from normal respiration and to quantitatively define the time to symptom onset and disease duration. A 0.85 rabbit intramuscular median lethal dose of BoNT/A elicited the most consistent and prolonged disease duration (mean = 11.8 days, relative standard deviation = 27.9%) that still enabled spontaneous recovery. Post-exposure treatment with antitoxin at various time points significantly shortened the disease duration, providing a proof of concept that the new model is adequate for evaluating novel therapeutics for botulism.

Botulinum neurotoxins (BoNTs) are the most poisonous substances in nature. Currently, the only therapy for botulism is antitoxin. This therapy suffers from several limitations and hence new therapeutic strategies are desired. One of the limitations in discovering BoNT inhibitors is the absence of an in vitro assay that correlates with toxin neutralization in vivo. In this work, a high-throughput screening assay for receptor-binding inhibitors against BoNT/A was developed. The assay is composed of two chimeric proteins: a receptor-simulating protein, consisting of the fourth luminal loop of synaptic vesicle protein 2C fused to glutathione-S-transferase, and a toxin-simulating protein, consisting of the receptor-binding domain of BoNT/A fused to beta-galactosidase. The assay was applied to screen the LOPAC1280 compound library. Seven selected compounds were evaluated in mice exposed to a lethal dose of BoNT/A. The compound aurintricarboxylic acid (ATA) conferred 92% protection, whereas significant delayed time to death ( p < 0.005) was observed for three additional compounds. Remarkably, ATA was also fully protective in mice challenged with a lethal dose of BoNT/E, which also uses the SV2 receptor. This study demonstrates that receptor-binding inhibitors have the potential to serve as next generation therapeutics for botulism, and therefore the assay developed may facilitate discovery of new anti-BoNT countermeasures.

Antitoxin is currently the only approved therapy for botulinum intoxications. The efficacy of antitoxin preparations is evaluated in animals. However, while in practice antitoxin is administered to patients only after symptom onset, in most animal studies, it is tested in relation to time postintoxication. This may be attributed to difficulties in quantitating early botulism symptoms in animals. In the current study, a novel system based on high-resolution monitoring of mouse activity on a running wheel was developed to allow evaluation of postsymptom antitoxin efficacy. The system enables automatic and remote monitoring of 48 mice simultaneously. Based on the nocturnal activity patterns of individual naive mice, two criteria were defined as the onset of symptoms. Postsymptom treatment with a human-normalized dose of antitoxin was fully protective in mice exposed to 4 50% lethal doses (LD 50 s) of botulinum neurotoxin serotype A (BoNT/A) and BoNT/B. Moreover, for the first time, a high protection rate was obtained in mice treated postsymptomatically, following a challenge with BoNT/E, the fastest-acting BoNT. The running wheel system was further modified to develop a mouse model for the evaluation of next-generation therapeutics for progressive botulism at time points where antitoxin is not effective. Exposure of mice to 0.3 LD 50 of BoNT/A resulted in long-lasting paralysis and a reduction in running activity for 16 to 18 days. Antitoxin treatment was no longer effective when administered 72 h postintoxication, defining the time window to evaluate next-generation therapeutics. Altogether, the running wheel systems presented herein offer quantitative means to evaluate the efficacy of current and future antibotulinum drugs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) global pandemic. The first step of viral infection is cell attachment, which is mediated by the binding of the SARS-CoV-2 receptor binding domain (RBD), part of the virus spike protein, to human angiotensin-converting enzyme 2 (ACE2). Therefore, drug repurposing to discover RBD-ACE2 binding inhibitors may provide a rapid and safe approach for COVID-19 therapy. Here, we describe the development of an in vitro RBD-ACE2 binding assay and its application to identify inhibitors of the interaction of the SARS-CoV-2 RBD to ACE2 by the high-throughput screening of two compound libraries (LOPAC ® 1280 and DiscoveryProbeTM). Three compounds, heparin sodium, aurintricarboxylic acid (ATA), and ellagic acid, were found to exert an effective binding inhibition, with IC50 values ranging from 0.6 to 5.5 µg/mL. A plaque reduction assay in Vero E6 cells infected with a SARS-CoV-2 surrogate virus confirmed the inhibition efficacy of heparin sodium and ATA. Molecular docking analysis located potential binding sites of these compounds in the RBD. In light of these findings, the screening system described herein can be applied to other drug libraries to discover potent SARS-CoV-2 inhibitors.

Medical treatment may require the continuous intravenous (IV) infusion of drugs to sustain the therapeutic blood concentration and to minimize dosing errors. Animal disease models that ultimately mimic the intended use of new potential drugs via a continuous IV infusion in unrestrained, free roaming animals are required. While peripherally inserted central catheters (PICCs) and other central line techniques for prolonged IV infusion of drugs are prevalent in the clinic, continuous IV infusion methods in an animal model are challenging and limited. In most cases, continuous IV infusion methods require surgical knowledge as well as expensive and complicated equipment. In the current work, we established a novel rabbit model for prolonged continuous IV infusion by inserting a PICC line from the marginal ear vein to the superior vena cava and connecting it to an externally carried ambulatory infusion pump. Either saline or a clinically relevant formulation could be steadily and continuously infused at 3-6 ml/h for 11 consecutive days into freely moving rabbits while maintaining normal body temperature, weight, and respiration physiology, as determined by daily spirometry. This new model is simple to execute and can advance the ability to administer and test new drug candidates., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Dor, David, Dekel Jaoui, Schwartz, Baruchi, Torgeman, Ben David, Rosen, Tal, Rosner, Zichel and Diamant.)

Botulinum neurotoxin (BoNT) serotypes A, B and E are responsible for most cases of human botulism. The only approved therapy for botulism is antitoxin treatment administered to patients after symptom onset. However, a recent meta-analysis of antitoxin efficacy in human botulism cases over the past century concluded that a statistically significant reduction in mortality is associated with the use of type E and type A antitoxin, but not with type B antitoxin. Animal models could be highly valuable in studying postsymptom antitoxin efficacy (PSAE). However, the few attempts to evaluate PSAE in animals relied on subjective observations and showed ∼50% protection. Recently, we developed a novel spirometry model for the quantitative evaluation of PSAE in rabbits and used it to demonstrate full protection against BoNT/E. In the current study, a comparative evaluation of PSAE in botulism types A and B was conducted using this quantitative respiratory model. A lethal dose of each toxin induced a comparable course of disease both in terms of time to symptoms (TTS, 41.9±1.3 and 40.6±1.1 h, respectively) and of time to death (TTD, 71.3±3.1 and 66.3±1.7 h, respectively). However, in accordance with the differential serotypic PSAE observed in humans, postsymptom antitoxin treatment was fully effective only in BoNT/A-intoxicated rabbits. This serotypic divergence was reflected by a positive and statistically significant correlation between TTS and TTD in BoNT/A-intoxicated rabbits (r=0.91, P =0.0006), but not in those intoxicated with BoNT/B (r=0.06, P =0.88). The rabbit spirometry system might be useful in the evaluation toolkit of botulism therapeutics, including those under development and intended to act when antitoxin is no longer effective., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Botulinum neurotoxins (BoNTs), the most poisonous substances known in nature, pose significant concern to health authorities. The only approved therapeutic for botulism is antitoxin. While administered to patients only after symptom onset, antitoxin efficacy is evaluated in animals mostly in relation to time postintoxication regardless of symptoms. This is most likely due to the difficulty in measuring early symptoms of botulism in animals. In this study, a rabbit spirometry model was developed to quantify early respiratory symptoms of type E botulism that were further used as a trigger for treatment. Impaired respiration, in the form of a reduced minute volume, was detected as early as 18.1 ± 2.9 h after intramuscular exposure to 2 rabbit 50% lethal doses (LD 50 ) of BoNT serotype E (BoNT/E), preceding any visible symptoms. All rabbits treated with antitoxin immediately following symptom onset survived. Postsymptom antitoxin efficacy was further evaluated in relation to toxin and antitoxin dosages as well as delayed antitoxin administration. Our system enabled us to demonstrate, for the first time, full antitoxin protection of animals treated with antitoxin after the onset of objective and quantitative type E botulism symptoms. This model may be utilized to evaluate the efficacy of antitoxins for additional serotypes of BoNT as well as that of next-generation anti-BoNT drugs that enter affected cells and act when antitoxin is no longer effective., (Copyright © 2018 American Society for Microbiology.)

Botulism therapy relies on passive immunization with antitoxin. The mouse neutralization test is the only pharmacopeia assay to measure the potency of antitoxin preparations. Herein, we present an in vitro cell-based assay for the measurement of pharmaceutical type A antitoxin potency. Accuracy, reproducibility and compatibility with the mouse bioassay were demonstrated using different batches of standard antitoxin and toxin preparations. The established assay may substantially reduce the use of laboratory animals in the process of pharmaceutical antitoxin production., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The only approved treatment for botulism relies on passive immunity which is mostly based on antibody preparations collected from hyper-immune horses. The IgG Fc fragment is commonly removed from these heterologous preparations to reduce the incidence of hyper-sensitivity reactions. New-generation therapies entering the pipeline are based on a combination of humanized monoclonal antibodies (MAbs), which exhibit improved safety and pharmacokinetics. In the current study, a systematic and quantitative approach was applied to measure the direct contribution of homologous Fc to the potency of monoclonal and polyclonal antitoxin preparations in mice. Homologous Fc increased the potency of three individual anti-botulinum toxin MAbs by up to one order of magnitude. Moreover, Fc fragment removal almost completely abolished the synergistic potency obtained from a combined preparation of these three MAbs. The MAb mixture neutralized a 400-mouse median lethal dose (MsLD50) of botulinum toxin, whereas the F(ab')2 combination failed to neutralize 10 MsLD50 of botulinum toxin. Notably, increased avidity did not compensate for this phenomenon, as a polyclonal, hyper-immune, homologous preparation lost 90% of its potency as well upon Fc removal. Finally, the addition of homologous Fc arms to a heterologous pharmaceutical anti-botulinum toxin polyclonal horse F(ab')2 preparation improved its efficacy when administered to intoxicated symptomatic mice. Our study extends the aspects by which switching from animal-based to human-based antitoxins will improve not only the safety but also the potency and efficacy of passive immunity against toxins.

Botulinum toxin was detected in patient serum using Endopeptidase-mass-spectrometry assay, although all conventional tests provided negative results. Antitoxin was administered, resulting in patient improvement. Implementing this highly sensitive and rapid assay will improve preparedness for foodborne botulism and deliberate exposure., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Monoclonal antibodies (MAbs) are among the fastest-growing therapeutics and are being developed for a broad range of indications, including the neutralization of toxins, bacteria and viruses. Nevertheless, MAbs potency is still relatively low when compared to conventional polyclonal Ab preparations. Moreover, the efficacy of an individual neutralizing MAb may significantly be hampered by the potential absence or modification of its target epitope in a mutant or subtype of the infectious agent. These limitations of individual neutralizing MAbs can be overcome by using oligoclonal combinations of several MAbs with different specificities to the target antigen. Studies conducted in our lab and by others show that such combined MAb preparation may present substantial synergy in its potency over the calculated additive potency of its individual MAb components. Moreover, oligoclonal preparation is expected to be better suited to compensating for reduced efficacy due to epitope variation. In this review, the synergistic neutralization properties of combined oligoclonal Ab preparations are described. The effect of Ab affinity, autologous Fc fraction, and targeting a critical number of epitopes, as well as the unexpected contribution of non-neutralizing clones to the synergistic neutralizing effect are presented and discussed.

Botulinum neurotoxins (BoNT) are considered some of the most lethal known substances. There are seven botulinum serotypes, of which types A, B and E cause most human botulism cases. Anti-botulinum polyclonal antibodies (PAbs) are currently used for both detection and treatment of the disease. However, significant improvements in immunoassay specificity and treatment safety may be made using monoclonal antibodies (MAbs). In this study, we present an approach for the simultaneous generation of highly specific and neutralizing MAbs against botulinum serotypes A, B, and E in a single process. The approach relies on immunization of mice with a trivalent mixture of recombinant C-terminal fragment (Hc) of each of the three neurotoxins, followed by a parallel differential robotic hybridoma screening. This strategy enabled the cloning of seven to nine MAbs against each serotype. The majority of the MAbs possessed higher anti-botulinum ELISA titers than anti-botulinum PAbs and had up to five orders of magnitude greater specificity. When tested for their potency in mice, neutralizing MAbs were obtained for all three serotypes and protected against toxin doses of 10 MsLD50-500 MsLD50. A strong synergistic effect of up to 400-fold enhancement in the neutralizing activity was observed when serotype-specific MAbs were combined. Furthermore, the highly protective oligoclonal combinations were as potent as a horse-derived PAb pharmaceutical preparation. Interestingly, MAbs that failed to demonstrate individual neutralizing activity were observed to make a significant contribution to the synergistic effect in the oligoclonal preparation. Together, the trivalent immunization strategy and differential screening approach enabled us to generate highly specific MAbs against each of the A, B, and E BoNTs. These new MAbs may possess diagnostic and therapeutic potential.

The receptor binding domain of botulinum neurotoxin (BoNT), also designated the C terminus of the heavy chain (H(C)), is a promising vaccine candidate against botulism. In this study, a highly efficient expression system for the protein was developed in Escherichia coli, which provided yields that were 1 order of magnitude higher than those reported to date (350 mg H(C) per liter). The product was highly immunogenic, protecting mice from a challenge with 10(5) 50% lethal dose (LD(50)) after a single vaccination and generating a neutralizing titer of 49.98 IU/ml after three immunizations. In addition, a single boost with HC increased neutralizing titers by up to 1 order of magnitude in rabbits hyperimmunized against toxoid. Moreover, we demonstrate here for the first time in vivo inhibition of BoNT/A intoxication by H(C)/A, presumably due to a blockade of the neurotoxin protein receptor SV2. Administration of HC/A delayed the time to death from 10.4 to 27.3 h in mice exposed to a lethal dose of BoNT/A (P = 0.0005). Since BoNT/A and BoNT/E partially share SV2 isoforms as their protein receptors, the ability of H(C)/A to cross-inhibit BoNT/E intoxication was evaluated. The administration of H(C)/A together with BoNT/E led to 50% survival and significantly delayed the time to death for the nonsurviving mice (P = 0.003). Furthermore, a combination of H(C)/A and a subprotective dose of antitoxin E fully protected mice against 850 mouse LD(50) of BoNT/E, suggesting complementary mechanisms of protection consisting of toxin neutralization by antibodies and receptor blocking by H(C)/A.

It is generally accepted that human vision is an extremely powerful information processing system that facilitates our interaction with the surrounding world. However, despite extended and extensive research efforts, which encompass many exploration fields, the underlying fundamentals and operational principles of visual information processing in human brain remain unknown. We still are unable to figure out where and how along the path from eyes to the cortex the sensory input perceived by the retina is converted into a meaningful object representation, which can be consciously manipulated by the brain. Studying the vast literature considering the various aspects of brain information processing, I was surprised to learn that the respected scholarly discussion is totally indifferent to the basic keynote question: "What is information?" in general or "What is visual information?" in particular. In the old days, it was assumed that any scientific research approach has first to define its basic departure points. Why was it overlooked in brain information processing research remains a conundrum. In this paper, I am trying to find a remedy for this bizarre situation. I propose an uncommon definition of "information", which can be derived from Kolmogorov's Complexity Theory and Chaitin's notion of Algorithmic Information. Embracing this new definition leads to an inevitable revision of traditional dogmas that shape the state of the art of brain information processing research. I hope this revision would better serve the challenging goal of human visual information processing modeling.

Identification and typing of spoilage and pathogenic microorganisms have become major objectives over the past decade in microbiology. In food, strain typing is necessary to ensure food safety and for linking cases of foodborne infections to suspected items. Recent advances in molecular biology have resulted in the development of numerous DNA-based methods for discrimination among bacterial strains. Here, we present the use of Simple Sequence Repeats (SSR, or Microsatellites) for bacterial typing. SSRs are a class of short DNA sequence motifs that are tandemly repeated at a specific locus. Computer-based screen of the complete genomic DNA sequences of various prokaryotes showed the existence of tens of thousands well distributed SSR tracts. Mono Nucleotides Repeats (MNRs) are the majority of SSR tracts in bacteria, therefore selected MNR loci were analyzed for variation among strains belonging to three bacterial species: Escherichia coli, Listeria monocytogenes and Vibrio cholerae. High levels of polymorphism in the number of repeats was observed. The finding that most of the MNR tracts are variable in bacterial genomes, but stable at the strain level, allows the use of MNRs for bacterial strains identification. The variation in MNR tracts enables the separation between virulent and non-virulent strain groups and further discriminates among bacterial isolates, in the three tested bacterial species. The uncovered MNR polymorphism is important as a genome-wide source of variation, both in practical applications (e.g. rapid strain identification) and in evolutionary studies. This multi-locus MNR strategy could be applied for high throughput bacterial typing by assigning an "identity number" for each strain based on MNR variations. The developed typing technology should include the fingerprint database for large bacterial strain collections and a high throughput scanner. This accurate and rapid tool can have a major role in decreasing the incidences of food-related outbreaks and will contribute to limit epidemics.

DNA-based methods are increasingly important for bacterial typing. The high number of polymorphic sites present among closely related bacterial genomes is the basis for the presented method. The method identifies multilocus genomic polymorphisms in intergenic regions termed AILP (amplified intergenic locus polymorphism). For each locus, a pair of unique PCR primers was designed to amplify an intergenic sequence from one open reading frame (ORF) to the adjacent ORF. Presence, absence, and size variation of the amplification products were identified and used as genetic markers for rapidly differentiating among strains. Polymorphism was evaluated using 18 AILP sites among 28 strains of Listeria monocytogenes and 6 strains of Listeria spp. and 30 AILP markers among 27 strains of Escherichia coli. Up to four alleles per locus were identified among Listeria strains, and up to six were identified among E. coli strains. In both species, more than half of the AILP sites revealed intraspecies polymorphism. The AILP data were applied to phylogenetic analysis among Listeria and E. coli strains. A clear distinction between L. monocytogenes and Listeria spp. was demonstrated. In addition, the method separated L. monocytogenes into the three known lineages and discriminated the most common virulent serotypic group, 4b. In E. coli, AILP analysis separated the known groups as well as the virulent O157:H7 isolates. These findings for both Listeria and E. coli are in agreement with other phylogenetic studies using molecular markers. The AILP method was found to be rapid, simple, reproducible, and a low-cost method for initial bacterial typing that could serve as a basis for epidemiological investigation.

Ligand-independent signals that are produced by the B-cell antigen receptor (BCR) confer an important positive selection checkpoint for immature B cells. Generation of inappropriate signals imposes developmental arrest of immature B cells, though the fate of these cells has not been investigated. Studies have shown that the lack of CD19 results in inappropriate signaling. In immunoglobulin transgenic mice, this inappropriate signaling impairs positive selection and stimulates receptor editing. Here, we studied the extent and significance of receptor editing in CD19-regulated positive selection of normal, nontransgenic B lymphopoiesis, using our bone marrow culture system. We found that the lack of CD19 resulted in elevated tonic signaling and impaired maturation, as revealed by surface marker expression and by functional assays. Immature CD19-/- B cells did not suppress RAG and underwent intensive receptor editing attempts in culture. Finally, in vivo analysis of light-chain isotype expression and Jkappa use in CD19-/- mice validated our in vitro observations. Our results suggest that CD19 has an important function in regulating positive selection and maturation of nontransgenic B-cell precursors and that receptor editing is an important salvage mechanism for immature B cells that fail positive selection.

Pathogenic autoantibodies detected in autoimmune diseases are predominantly IgG isotypes, reflecting the generation and activation of an autoimmune memory B cell repertoire. It is not completely understood how such autoreactive cells are generated and escape central and/or peripheral tolerance mechanisms, and several models to explain this have been proposed. It is generally thought that B lymphocytes utilize IgM receptors for development and tolerance establishment, whereas IgG receptors are primarily used to promote memory formation and signal for memory-type responses. In here we review recent findings suggesting that spontaneously occurring class switch recombination in B lymphopoiesis confer B lymphocytes with a novel developmental pathway that is driven by non-IgM receptors. The physiological relevance of this developmental pathway in generating an autoimmune memory repertoire, as well as a Fas-dependent mechanism regulating it, is discussed.

Maturation of B lymphocytes strictly depends on the signaling competence of the B cell antigen receptor (BCR). Autoreactive receptors undergo negative selection and can be replaced by receptor editing. In addition, the process of maturation of non-self B cells and migration to the spleen, referred to as positive selection, is limited by the signaling competence of the BCR. Using 3-83Tg mice deficient of CD19 we have shown that signaling incompetence not only blocks positive selection but also activates receptor editing. Here we study the role of ligand-independent BCR tonic tyrosine phosphorylation signals in activation of receptor editing. We find that editing, immature 3-83Tg B cells deficient of CD19 have elevated BCR tonic signals and that lowering these tonic signals effectively suppresses receptor editing. Furthermore, we show that elevation of BCR tonic signals in non-editing, immature 3-83Tg B cells stimulates significant receptor editing. We also show that positive selection and developmental progression from the bone marrow to the spleen are limited to cells capable of establishing appropriate tonic signals, as in contrast to immature cells, splenic 3-83Tg B cells deficient of CD19 have BCR tonic signals similar to those of the control 3-83Tg cells. This developmental progression is accompanied by activation of molecules signaling for growth and survival. Hence, we suggest that ligand-independent BCR tonic signals are required for promoting positive selection and suppressing the receptor-editing mechanism in immature B cells.

Multilocus sequencing of housekeeping genes has been used previously for bacterial strain typing and for inferring evolutionary relationships among strains of Escherichia coli. In this study, we used shorter intergenic sequences that contained simple sequence repeats (SSRs) of repeating mononucleotide motifs (mononucleotide repeats [MNRs]) to infer the phylogeny of pathogenic and commensal E. coli strains. Seven noncoding loci (four MNRs and three non-SSRs) were sequenced in 27 strains, including enterohemorrhagic (six isolates of O157:H7), enteropathogenic, enterotoxigenic, B, and K-12 strains. The four MNRs were also sequenced in 20 representative strains of the E. coli reference (ECOR) collection. Sequence polymorphism was significantly higher at the MNR loci, including the flanking sequences, indicating a higher mutation rate in the sequences flanking the MNR tracts. The four MNR loci were amplifiable by PCR in the standard ECOR A, B1, and D groups, but only one (yaiN) in the B2 group was amplified, which is consistent with previous studies that suggested that B2 is the most ancient group. High sequence compatibility was found between the four MNR loci, indicating that they are in the same clonal frame. The phylogenetic trees that were constructed from the sequence data were in good agreement with those of previous studies that used multilocus enzyme electrophoresis. The results demonstrate that MNR loci are useful for inferring phylogenetic relationships and provide much higher sequence variation than housekeeping genes. Therefore, the use of MNR loci for multilocus sequence typing should prove efficient for clinical diagnostics, epidemiology, and evolutionary study of bacteria.