Human red blood cell acetylcholinesterase activity: a revisit after fifteen years.

Human red blood cell acetylcholinesterase (RBC-AChE) activity is valuable for detecting potential exposure to cholinesterase inhibiting substances (CIS). A reliable population-based RBC-AChE activity reference range is critical for early and massive clinical and occupational toxicology screening. Previous published studies were often limited to small numbers of subjects, various testing methods, and crude statistical data analyses. We tested 4818 adult subjects with a well-established 17-minute modified Michel method over a 2-year period. We conducted a retrospective data analysis and systematically investigated on the influences to testing values from gender, age, age group, and their combinations and interactions. No significant difference was observed in the testing values between males (mean, medium, interquartile range = 0.76, 0.76, 0.71-0.80 ΔpH/h, respectively) and females (mean, medium, interquartile range = 0.76, 0.76, 0.71-0.81 ΔpH/hour, respectively), when gender was the only factor considered (p = 0.7238). However, with age progression, male testing values exhibited a consistent upward trend, while females did not show any clear patterns. Linear regression analysis of the data revealed that gender, age, and age group more or less affected testing values either as independent variables or with their combinations and interactions. However, more potential factors need to be included to achieve better testing value predictions. We recommend the toxicological testing community to adopt a new set of age group specific RBC-AChE activity reference ranges for males (0.68-0.80, 0.69-0.81, 0.70-0.83, 0.71-0.84, and 0.73-0.87 ΔpH/h for 18-29, 30-39, 40-49, 50-59, and ≥60 years old, respectively) while keeping the current reference range (0.63-0.89 ΔpH/hour) for females.

The COVID-19 pandemic: A time for veterinary leadership in one health.

The COVID-19 pandemic is a wake-up call for leaders of the North American veterinary profession. Veterinary professionals constitute an invaluable workforce capable of delivering public services essential to weathering the current pandemic and preventing future pandemics. Yet North American veterinary professionals are often underutilized in efforts that could maximize their contributions to public health and research. Misguided public perception about the utility of the profession might hinder One Health partnerships between veterinary professionals and diverse collaborators. Society in general demands that leadership across several levels of the profession expand and diversify efforts aimed at greater public service while preserving societal benefits stemming from companion animal care. The pandemic's most deeply rooted effects could lie in its potential degradation of global food security, which can be better preserved if more veterinarians embrace their public health foundations. The pandemic is an opportunity and obligation for change. Failing to seize this moment could undermine public health and global security for generations.

Streptococcus bovis/S. equinus complex septicemia in a group of calves following intramuscular vaccination.

Organisms previously classified as Streptococcus bovis (i.e., the S. bovis/S. equinus complex) are common in cattle feces, but may also act as opportunistic pathogens. In the current work, Streptococcus infantarius subsp. coli, a member of this complex, was associated of a cluster of calves that died within hours of injection with a modified live viral vaccine. Within 12 h of vaccination of 46 calves at a cow/calf operation, 4 calves had died, 3 calves were ill, and 1 unvaccinated cow was dead. Autopsies were performed on the cow, 2 dead calves, and 1 affected surviving calf, which was euthanized ~24 h after vaccine administration. The animals had similar gross anatomic and microscopic lesions, including subcutaneous and intramuscular dark hemorrhage on the caudal neck, multiorgan ecchymosis and petechiation, and alveolitis to interstitial pneumonia. Gram-positive cocci were in the vasculature of the lung and skeletal muscle, and S. infantarius subsp. coli was cultured from tissues and from the vaccines used on affected animals, but not in vials used on unaffected animals. Together, these findings suggest death caused by streptococcal septicemia and toxemia as a result of contamination.

Characterization of coding synonymous and non-synonymous variants in ADAMTS13 using ex vivo and in silico approaches.

Synonymous variations, which are defined as codon substitutions that do not change the encoded amino acid, were previously thought to have no effect on the properties of the synthesized protein(s). However, mounting evidence shows that these "silent" variations can have a significant impact on protein expression and function and should no longer be considered "silent". Here, the effects of six synonymous and six non-synonymous variations, previously found in the gene of ADAMTS13, the von Willebrand Factor (VWF) cleaving hemostatic protease, have been investigated using a variety of approaches. The ADAMTS13 mRNA and protein expression levels, as well as the conformation and activity of the variants have been compared to that of wild-type ADAMTS13. Interestingly, not only the non-synonymous variants but also the synonymous variants have been found to change the protein expression levels, conformation and function. Bioinformatic analysis of ADAMTS13 mRNA structure, amino acid conservation and codon usage allowed us to establish correlations between mRNA stability, RSCU, and intracellular protein expression. This study demonstrates that variants and more specifically, synonymous variants can have a substantial and definite effect on ADAMTS13 function and that bioinformatic analysis may allow development of predictive tools to identify variants that will have significant effects on the encoded protein.

Detection of a secreted metalloprotease within the nuclei of liver cells.

ADAMTS13 is a secreted zinc metalloprotease expressed by various cell types. Here, we investigate its cellular pathway in endogenously expressing liver cell lines and after transient transfection with ADAMTS13. Besides compartmentalizations of the cellular secretory system, we detected an appreciable level of endogenous ADAMTS13 within the nucleus. A positively charged amino acid cluster (R-Q-R-Q-R-Q-R-R) present in the ADAMTS13 propeptide may act as a nuclear localization signal (NLS). Fusing this NLS-containing region to eGFP greatly potentiated its nuclear localization. Bioinformatics analysis suggests that the ADAMTS13 CUB-2 domain has a double-stranded beta helix (DSBH) structural architecture characteristic of various protein-protein interaction modules like nucleoplasmins, class I collagenase, tumor necrosis factor ligand superfamily, supernatant protein factor (SPF) and the B1 domain of neuropilin-2. Based on this contextual evidence and that largely conserved polar residues could be mapped on to a template CUB domain homolog, we hypothesize that a region in the ADAMTS13 CUB-2 domain with conserved polar residues might be involved in protein-protein interaction within the nucleus.

Characterization of conformation-sensitive antibodies to ADAMTS13, the von Willebrand cleavage protease.

BACKGROUND: The zinc metalloprotease ADAMTS13 is a multidomain protein that cleaves von Willebrand Factor (VWF) and is implicated in Thrombotic Thrombocytopenic Purpura (TTP) pathogenesis. Understanding the mechanism of this protein is an important goal. Conformation sensitive antibodies have been used to monitor protein conformation and to decipher the molecular mechanism of proteins as well as to distinguish functional and non-functional mutants. METHODOLOGY/PRINCIPAL FINDINGS: We have characterized several antibodies against ADAMTS13, both monoclonal and polyclonal. We have used flow cytometry to estimate the binding of these antibodies to ADAMTS13 and demonstrate that antibodies raised against the TSP and disintegrin domains detect conformation changes in the ADAMTS13. Thus for example, increased binding of these antibodies was detected in the presence of the substrate (VWF), mainly at 37 degrees C and not at 4 degrees C. These antibodies could also detect differences between wild-type ADAMTS13 and the catalytically deficient mutant (P475S). The flow cytometry approach also allows us to estimate the reactivity of the antibody as well as its apparent affinity. CONCLUSIONS/SIGNIFICANCE: Our results suggest that these antibodies may serve as useful reagents to distinguish functional and non-functional ADAMTS13 and analyze conformational transitions to understand the catalytic mechanism.

The development of gene therapy: from monogenic recessive disorders to complex diseases such as cancer.

During the last 4 decades, gene therapy has moved from preclinical to clinical studies for many diseases ranging from monogenic recessive disorders such as hemophilia to more complex diseases such as cancer, cardiovascular disorders, and human immunodeficiency virus (HIV). To date, more than 1,340 gene therapy clinical trials have been completed, are ongoing, or have been approved in 28 countries, using more than 100 genes. Most of those clinical trials (66.5%) were aimed at the treatment of cancer. Early hype, failures, and tragic events have now largely been replaced by the necessary stepwise progress needed to realize clinical benefits. We now understand better the strengths and weaknesses of various gene transfer vectors; this facilitates the choice of appropriate vectors for individual diseases. Continuous advances in our understanding of tumor biology have allowed the development of elegant, more efficient, and less toxic treatment strategies. In this introductory chapter, we review the history of gene therapy since the early 1960s and present in detail two major recurring themes in gene therapy: (1) the development of vector and delivery systems and (2) the design of strategies to fight or cure particular diseases. The field of cancer gene therapy experienced an "awkward adolescence." Although this field has certainly not yet reached maturity, it still holds the potential of alleviating the suffering of many individuals with cancer.

Patterning of multiple layered projections to the auditory midbrain prior to experience.

The precise arrangement of patterned inputs into discrete functional domains is a common organizational feature of primary sensory structures. While the specific organization of patterned connections has been well documented in the visual and somatosensory systems, comparatively little is known about the arrangement of neighboring afferent patterns in the emerging auditory system. Here we report early projection specificity for multiple converging inputs to the rat central nucleus of the inferior colliculus (ICC). Afferents arising from the dorsal cochlear nucleus (DCN), the dorsal nucleus of the lateral lemniscus (DNLL), and the lateral superior olive (LSO) establish discernible axonal layers a week prior to experience. By hearing onset, contralateral DCN and contralateral LSO layers are clearly defined and segregated from contralateral DNLL terminal zones. Layering of the ipsilateral LSO projection, on the other hand, exhibits considerable spatial overlap with the contralateral DNLL pattern. This fine laminar structure of interdigitating and overlapping inputs likely underlies the complex signal processing performed in the auditory midbrain and may serve as a model system for examining competitive interactions between neighboring excitatory and inhibitory projections early in development.