Electrophysiological Activity of Primary Cortical Neuron-Glia Mixed Cultures.

Neuroinflammation plays a central role in many neurological disorders, ranging from traumatic brain injuries to neurodegeneration. Electrophysiological activity is an essential measure of neuronal function, which is influenced by neuroinflammation. In order to study neuroinflammation and its electrophysiological fingerprints, there is a need for in vitro models that accurately capture the in vivo phenomena. In this study, we employed a new tri-culture of primary rat neurons, astrocytes, and microglia in combination with extracellular electrophysiological recording techniques using multiple electrode arrays (MEAs) to determine the effect of microglia on neural function and the response to neuroinflammatory stimuli. Specifically, we established the tri-culture and its corresponding neuron-astrocyte co-culture (lacking microglia) counterpart on custom MEAs and monitored their electrophysiological activity for 21 days to assess culture maturation and network formation. As a complementary assessment, we quantified synaptic puncta and averaged spike waveforms to determine the difference in excitatory to inhibitory neuron ratio (E/I ratio) of the neurons. The results demonstrate that the microglia in the tri-culture do not disrupt neural network formation and stability and may be a better representation of the in vivo rat cortex due to its more similar E/I ratio as compared to more traditional isolated neuron and neuron-astrocyte co-cultures. In addition, only the tri-culture displayed a significant decrease in both the number of active channels and spike frequency following pro-inflammatory lipopolysaccharide exposure, highlighting the critical role of microglia in capturing electrophysiological manifestations of a representative neuroinflammatory insult. We expect the demonstrated technology to assist in studying various brain disease mechanisms.

Botulinum Neurotoxin A4 Has a 1000-Fold Reduced Potency Due to Three Single Amino Acid Alterations in the Protein Receptor Binding Domain.

Botulinum neurotoxin subtype A4 (BoNT/A4) is ~1000-fold less potent than BoNT/A1. This study addresses the basis for low BoNT/A4 potency. Utilizing BoNT/A1-A4 and BoNT/A4-A1 Light Chain-Heavy Chain (LC-HC) chimeras, HC-A4 was responsible for low BoNT/A4 potency. Earlier studies showed BoNT/A1-receptor binding domain (Hcc) bound a ß-strand peptide (556-564) and glycan-N559 within Luminal Domain 4 (LD4) of SV2C, the BoNT/A protein receptor. Relative to BoNT/A1, the Hcc of BoNT/A4 possesses two amino acid variants (D1141 and N1142) within the ß-peptide binding interface and one amino acid variant (R1292) located near the SV2C glycan-N559. Introduction of BoNT/A4 ß-strand peptide variant (D1141 and N1142) into BoNT/A1 reduced toxin potency 30-fold, and additional introduction of the BoNT/A4 glycan-N559 variant (D1141, N1142, and R1292) further reduced toxin potency to approach BoNT/A4. While introduction of BoNT/A1 glycan-N559 variant (G1292) into BoNT/A4 did not alter toxin potency, additional introduction of BoNT/A1 ß-strand peptide variants (G1141, S1142, and G1292) resulted in potency approaching BoNT/A1 potency. Thus, outcomes from these functional and modeling studies indicate that in rodent models, disruption of Hcc -SV2C ß-peptide and -glycan-N559 interactions mediate low BoNT/A4 potency, while in human motor neurons, disruption of Hcc-SV2C ß-peptide alone mediates low BoNT/A4 potency, which link to a species-specific variation at SV2C563.

How Botulinum Neurotoxin Light Chain A1 Maintains Stable Association with the Intracellular Neuronal Plasma Membrane.

Botulinum neurotoxin serotype A (BoNT/A) is the most potent protein toxin for humans and is utilized as a therapy for numerous neurologic diseases. BoNT/A comprises a catalytic Light Chain (LC/A) and a Heavy Chain (HC/A) and includes eight subtypes (BoNT/A1-/A8). Previously we showed BoNT/A potency positively correlated with stable localization on the intracellular plasma membrane and identified a low homology domain (amino acids 268-357) responsible for LC/A1 stable co-localization with SNAP-25 on the plasma membrane, while LC/A3 was present in the cytosol of Neuro2A cells. In the present study, steady-state- and live-imaging of a cytosolic LC/A3 derivative (LC/A3V) engineered to contain individual structural elements of the A1 LDH showed that a 59 amino acid region (275-334) termed the MLD was sufficient to direct LC/A3V from the cytosol to the plasma membrane co-localized with SNAP-25. Informatics and experimental validation of the MLD-predicted R1 region (an α-helix, residues 275-300) and R2 region (a loop, α-helix, loop, residues 302-334) both contribute independent steps to the stable co-localization of LC/A1 with SNAP-25 on the plasma membrane of Neuro-2A cells. Understanding how these structural elements contribute to the overall association of LC/A1 on the plasma membrane may identify the molecular basis for the LC contribution of BoNT/A1 to high potency.

Influence of microchannel geometry on device performance and electrophysiological recording fidelity during long-term studies of connected neural populations.

Compartmentalized microfluidic neural cell culture platforms, which physically separate axons from the neural soma using a series of microchannels, have been used for studying a wide range of pathological conditions and basic neuroscience questions. While each study has different experimental needs, the fundamental design of these devices has largely remained unchanged and a systematic study to establish long-term neural cultures in this format is lacking. Here, we investigate the influence of microchannel geometry and cell seeding density on device performance particularly in the context of long-term studies of synaptically-connected, yet fluidically-isolated neural populations of neurons and glia. Of the different experimental parameters, the microchannel height was the principal determinant of device performance, where the other parameters offer additional degrees of freedom in customizing such devices for specific applications. We condense the effects of these parameters into design rules and demonstrate their utility in engineering a microfluidic neural culture platform with integrated microelectrode arrays. The engineered device successfully recorded from primary rat cortical cells for 59 days in vitro with more than on order of magnitude enhancement in signal-to-noise ratio in the microchannels.

COVID-19 Induced Taste Dysfunction and Recovery: Association with Smell Dysfunction and Oral Health Behaviour.

Background and Objectives: Disruption to taste and smell are common symptoms of COVID-19 infection. The current literature overlooks taste symptoms and tends to focus on the sense of smell. Persisting cases (>28 days) of taste dysfunction are increasingly recognised as a major future healthcare challenge. This study focuses on the severity and recovery of COVID-19 induced taste loss and association with olfactory symptoms, lifestyle and oral health factors. Materials and Methods: This study was a cross-sectional survey comparing 182 rapid taste recovery participants (≤28 days) with 47 participants with prolonged taste recovery >28 days. Analyses of taste loss in association with smell loss, age, sex, illness severity, diet, BMI, vitamin-D supplementation, antidepressants, alcohol use, smoking, brushing frequency, flossing, missing teeth, appliances and number of dental restorations were conducted. Differences in the severity of the loss of sour, sweet, salt, bitter and umami tastes were explored. Results: Both the severity and the duration of taste and smell loss were closely correlated (p < 0.001). Salt taste was significantly less affected than all other taste qualities (p < 0.001). Persisting taste loss was associated with older age (mean ± 95% CI = 31.73 ± 1.23 years vs. 36.66 ± 3.59 years, p < 0.001) and reduced likelihood of using floss (odds ratio ± 95% CI = 2.22 (1.15−4.25), p = 0.047). Conclusions: Smell and taste loss in COVID-19 are closely related, although a minority of individuals can experience taste or smell dysfunction in the absence of the other. The taste of salt may be less severely affected than other taste qualities and future work exploring this finding objectively is indicated. The association of flossing with rapid taste recovery adds to the growing evidence of a link between good periodontal health and favourable COVID-19 outcomes.

Relationship between Recovery from COVID-19-Induced Smell Loss and General and Oral Health Factors.

Background and Objectives: Loss of smell is one of the strongest predictors of coronavirus disease 2019 (COVID-19) and can persist long after other symptoms have resolved. "Long" cases (>28 days) of smell dysfunction present future challenges to medical and dental professionals, as there is a lack of evidence on the causes and any exacerbating or relieving factors. This study aimed to explore the persistence of COVID-19-induced smell loss and association with physical, lifestyle and oral health factors. Materials and Methods: This study was a cross-sectional survey of 235 participants. Recovery of smell was explored, comparing rapid recovery (≤28 days) with prolonged recovery (>28 days). Associative factors included age, sex, illness severity, diet, BMI, vitamin D supplementation, antidepressants, alcohol use, smoking, brushing frequency, flossing, missing teeth, appliances and number of dental restorations. Results: Smell loss showed 87% resolution within 30 days. Prolonged smell loss was significantly associated with older age (mean ± 95%, CI = 31.53 ± 1.36 years for rapid recovery vs. mean ± 95%, CI = 36.0 ± 3 years for prolonged recovery, p = 0.003) and increased self-reported illness severity (mean ± 95%, CI = 4.39 ± 0.27 for rapid recovery vs. 5.01 ± 0.54 for prolonged recovery, p = 0.016). Fisher's exact test revealed flossing was associated with rapid recovery, with flossers comprising 75% of the rapid-recovery group, compared to 56% in the prolonged-recovery group (odds ratio ± 95%, CI = 2.26 (1.23-4.15), p = 0.01). All other factors were not significantly associated (p > 0.05). Conclusions: Increased age and illness severity were associated with prolonged smell recovery. Use of floss was the only modifiable factor associated with rapid recovery of smell loss. As 87% of cases resolve within 30 days, future studies may benefit from targeted recruitment of individuals experiencing prolonged sense loss. This would increase statistical confidence when declaring no association with the other factors assessed, avoiding type II errors.

Resolution of Two Steps in Botulinum Neurotoxin Serotype A1 Light Chain Localization to the Intracellular Plasma Membrane.

Botulinum neurotoxin serotype A (BoNT/A) is the most potent protein toxin to humans. BoNT/A light chain (LC/A) cleavage of the membrane-bound SNAP-25 has been well-characterized, but how LC/A traffics to the plasma membrane to target SNAP-25 is unknown. Of the eight BoNT/A subtypes (A1-A8), LC/A3 has a unique short duration of action and low potency that correlate to the intracellular steady state of LC/A, where LC/A1 is associated with the plasma membrane and LC/A3 is present in the cytosol. Steady-state and live imaging of LC/A3-A1 chimeras identified a two-step process where the LC/A N terminus bound intracellular vesicles, which facilitated an internal α-helical-rich domain to mediate LC/A plasma membrane association. The propensity of LC/A variants for membrane association correlated with enhanced BoNT/A potency. Understanding the basis for light chain intracellular localization provides insight to mechanisms underlying BoNT/A potency, which can be extended to applications as a human therapy.

Non-invasive Urinary Biomarkers in Moyamoya Disease.

Introduction: A major difficulty in treating moyamoya disease is the lack of effective methods to detect novel or progressive disease prior to the onset of disabling stroke. More importantly, a tool to better stratify operative candidates and quantify response to therapy could substantively complement existing methods. Here, we present proof-of-principle data supporting the use of urinary biomarkers as diagnostic adjuncts in pediatric moyamoya patients. Methods: Urine and cerebrospinal fluid specimens were collected from pediatric patients with moyamoya disease and a cohort of age and sex-matched control patients. Clinical and radiographic data were paired with measurements of a previously validated panel of angiogenic proteins quantified by ELISA. Results were compared to age and sex-matched controls and subjected to statistical analyses. Results: Evaluation of a specific panel of urinary and cerebrospinal fluid biomarkers by ELISA demonstrated significant elevations of angiogenic proteins in samples from moyamoya patients compared to matched controls. ROC curves for individual urinary biomarkers, including MMP-2, MMP-9, MMP-9/NGAL, and VEGF, showed excellent discrimination. The optimal urinary biomarker was MMP-2, providing a sensitivity of 88%, specificity of 100%, and overall accuracy of 91%. Biomarker levels changed in response to therapy and correlated with radiographic evidence of revascularization. Conclusions: We report, for the first time, identification of a panel of urinary biomarkers that predicts the presence of moyamoya disease. These biomarkers correlate with presence of disease and can be tracked from the central nervous system to urine. These data support the hypothesis that urinary proteins are useful predictors of the presence of moyamoya disease and may provide a basis for a novel, non-invasive method to identify new disease and monitor known patients following treatment.

Comparison of machine learning algorithms for the prediction of five-year survival in oral squamous cell carcinoma.

BACKGROUND/AIM: Machine learning analyses of cancer outcomes for oral cancer remain sparse compared to other types of cancer like breast or lung. The purpose of the present study was to compare the performance of machine learning algorithms in the prediction of global, recurrence-free five-year survival in oral cancer patients based on clinical and histopathological data. METHODS: Data were gathered retrospectively from 416 patients with oral squamous cell carcinoma. The data set was divided into training and test data set (75:25 split). Training performance of five machine learning algorithms (Logistic regression, K-nearest neighbours, Naïve Bayes, Decision tree and Random forest classifiers) for prediction was assessed by k-fold cross-validation. Variables used in the machine learning models were age, sex, pain symptoms, grade of lesion, lymphovascular invasion, extracapsular extension, perineural invasion, bone invasion and type of treatment. Variable importance was assessed and model performance on the testing data was assessed using receiver operating characteristic curves, accuracy, sensitivity, specificity and F1 score. RESULTS: The best performing model was the Decision tree classifier, followed by the Logistic Regression model (accuracy 76% and 60%, respectively). The Naïve Bayes model did not display any predictive value with 0% specificity. CONCLUSIONS: Machine learning presents a promising and accessible toolset for improving prediction of oral cancer outcomes. Our findings add to a growing body of evidence that Decision tree models are useful in models in predicting OSCC outcomes. We would advise that future similar studies explore a variety of machine learning models including Logistic regression to help evaluate model performance.

Dysregulation of the EphrinB2-EphB4 ratio in pediatric cerebral arteriovenous malformations is associated with endothelial cell dysfunction in vitro and functions as a novel noninvasive biomarker in patients.

We investigated (1) EphrinB2 and EphB4 receptor expression in cerebral AVMs, (2) the impact of an altered EphrinB2:EphB4 ratio on brain endothelial cell function and (3) potential translational applications of these data. The following parameters were compared between AVM endothelial cells (AVMECs) and human brain microvascular endothelial cells (HBMVECs): quantified EphrinB2 and EphB4 expression, angiogenic potential, and responses to manipulation of the EphrinB2:EphB4 ratio via pharmacologic stimulation/inhibition. To investigate the clinical relevance of these in vitro data, Ephrin expression was assessed in AVM tissue (by immunohistochemistry) and urine (by ELISA) from pediatric patients with AVM (n = 30), other cerebrovascular disease (n = 14) and control patients (n = 29), and the data were subjected to univariate and multivariate statistical analyses. Compared to HBMVECs, AVMECs demonstrated increased invasion (p = 0.04) and migration (p = 0.08), impaired tube formation (p = 0.06) and increased EphrinB2:EphB4 ratios. Altering the EphrinB2:EphB4 ratio (by increasing EphrinB2 or blocking EphB4) in HBMVECs increased invasion (p = 0.03 and p < 0.05, respectively). EphrinB2 expression was increased in AVM tissue, which correlated with increased urinary EphrinB2 levels in AVM patients. Using the optimal urinary cutoff value (EphrinB2 > 25.7 pg/µg), AVMs were detected with high accuracy (80% vs. controls) and were distinguished from other cerebrovascular disease (75% accuracy). Post-treatment urinary EphrinB2 levels normalized in an index patient. In summary, AVMECs have an EphrinB2:EphB4 ratio that is increased compared to that of normal HBMVECs. Changing this ratio in HBMVECs induces AVMEC-like behavior. EphrinB2 is clinically relevant, and its levels are increased in AVM tissue and patient urine. This work suggests that dysregulation of the EphrinB2:EphB4 signaling cascade and increases in EphrinB2 may play a role in AVM development, with potential utility as a diagnostic and therapeutic target.

Salivary Metabolomics: From Diagnostic Biomarker Discovery to Investigating Biological Function.

Metabolomic profiling of biofluids, e.g., urine, plasma, has generated vast and ever-increasing amounts of knowledge over the last few decades. Paradoxically, metabolomic analysis of saliva, the most readily-available human biofluid, has lagged. This review explores the history of saliva-based metabolomics and summarizes current knowledge of salivary metabolomics. Current applications of salivary metabolomics have largely focused on diagnostic biomarker discovery and the diagnostic value of the current literature base is explored. There is also a small, albeit promising, literature base concerning the use of salivary metabolomics in monitoring athletic performance. Functional roles of salivary metabolites remain largely unexplored. Areas of emerging knowledge include the role of oral host-microbiome interactions in shaping the salivary metabolite profile and the potential roles of salivary metabolites in oral physiology, e.g., in taste perception. Discussion of future research directions describes the need to begin acquiring a greater knowledge of the function of salivary metabolites, a current research direction in the field of the gut metabolome. The role of saliva as an easily obtainable, information-rich fluid that could complement other gastrointestinal fluids in the exploration of the gut metabolome is emphasized.

Adhesive Hydrogels for Maxillofacial Tissue Regeneration Using Minimally Invasive Procedures.

Minimally invasive surgical procedures aiming to repair damaged maxillofacial tissues are hampered by its small, complex structures and difficult surgical access. Indeed, while arthroscopic procedures that deliver regenerative materials and/or cells are common in articulating joints such as the knee, there are currently no treatments that surgically place cells, regenerative factors or materials into maxillofacial tissues to foster bone, cartilage or muscle repair. Here, hyaluronic acid (HA)-based hydrogels are developed, which are suitable for use in minimally invasive procedures, that can adhere to the surrounding tissue, and deliver cells and potentially drugs. By modifying HA with both methacrylate (MA) and 3,4-dihydroxyphenylalanine (Dopa) groups using a completely aqueous synthesis route, it is shown that MA-HA-Dopa hydrogels can be applied under aqueous conditions, gel quickly using a standard surgical light, and adhere to tissue. Moreover, upon oxidation of the Dopa, human marrow stromal cells attach to hydrogels and survive when encapsulated within them. These observations show that when incorporated into HA-based hydrogels, Dopa moieties can foster cell and tissue interactions, ensuring surgical placement and potentially enabling delivery/recruitment of regenerative cells. The findings suggest that MA-HA-Dopa hydrogels may find use in minimally invasive procedures to foster maxillofacial tissue repair.

Endogenous salivary citrate is associated with enhanced rheological properties following oral capsaicin stimulation.

NEW FINDINGS: What is the central question of this study? What are the relationships between physical properties of saliva, protein composition and metabolite composition? What is the main finding and its importance? Salivary citrate, one of the major endogenous metabolites in saliva, increased upon capsaicin stimulation and was associated with improved physical properties measured by extensional rheology. This suggests salivary gland citrate transporters might be a valuable area of future study. ABSTRACT: Saliva displays viscoelastic properties which enable coating, lubrication and protection of the oral mucosa and hard tissues. Individuals lacking saliva or perceiving oral dryness can manage their symptoms using artificial saliva preparations, but these often fail to mimic the sensation and functionality of natural saliva. It is widely acknowledged that mucins (MUC7 and MUC5B) confer saliva's rheological properties, but artificial saliva containing purified mucins is still often an inadequate substitute. This work aimed to explore salivary components that influence salivary extensional rheology to better understand how natural saliva could be replicated. Saliva was stimulated via control and capsaicin solutions in healthy volunteers. Extensional rheology was analysed using a CaBER-1 (capillary breakup) extensional rheometer. Protein composition, including mucins, was measured by gel-electrophoresis band densitometry and metabolites were measured by 1 H nuclear magnetic resonance spectroscopy. Capsaicin stimulation significantly increased capillary breakup time, extensional viscosity and the abundance of most major salivary proteins. Stimulation also increased salivary citrate and choline concentrations. Significant correlations were found between capillary breakup time and amylase (r = 0.67, P < 0.05), statherin (ρ = 0.66, P < 0.05) and citrate (ρ = 0.81, P < 0.01). The relationship between citrate and salivary rheology was subsequently investigated in vitro. These results suggest that citrate and non-mucin proteins are stronger predictors of salivary rheology than the more often studied mucin glycoproteins. Potential mechanisms are discussed and future work in this area could help formulate more effective saliva substitutes, more closely resembling natural saliva.

Quantitative Analysis of Different Cell Entry Routes of Actively Targeted Nanomedicines Using Imaging Flow Cytometry.

A rapid, high-throughput, and quantitative method for cell entry route characterization is still lacking in nanomedicine research. Here, we report the application of imaging flow cytometry for quantitatively analyzing cell entry routes of actively targeted nanomedicines. We first engineered ICAM1 antibody-directed fusogenic nanoliposomes (ICAM1-FusoNLPs) and ICAM1 antibody-directed endocytic nanolipogels (ICAM1-EndoNLGs) featuring highly similar surface properties but different cell entry routes: receptor-mediated membrane fusion and receptor-mediated endocytosis, respectively. By using imaging flow cytometry, we characterized their intracellular delivery into human breast cancer MDA-MB-231 cells. We found that ICAM1-FusoNLPs mediated a 2.8-fold increased cell uptake of fluorescent payload, FITC-dextran, with a 2.4-fold increased intracellular distribution area in comparison with ICAM1-EndoNLGs. We also investigated the effects of incubation time and endocytic inhibitors on the cell entry routes of ICAM1-FusoNLP and ICAM1-EndoNLG. Our results indicate that receptor-mediated membrane fusion is a faster and more efficient cell entry route than receptor-mediated endocytosis, bringing with it a significant therapeutic benefit in a proof-of-principle nanomedicine-mediated siRNA transfection experiment. Our studies suggest that cell entry route may be an important design parameter to be considered in the development of next-generation nanomedicines. © 2019 International Society for Advancement of Cytometry.

Anatomical stability of human fungiform papillae and relationship with oral perception measured by salivary response and intensity rating.

Fungiform papillae house taste buds on the anterior dorsal tongue. Literature is inconclusive as to whether taste perception correlates with fungiform papillae density (FPD). Gustatory reflexes modulate the amount and composition of saliva subsequently produced, and thus may be a more physiologically objective measure of tastant-receptor interactions. Taste perception fluctuates with time but the stability of individual fungiform papillae is unclear. This study followed ten healthy volunteers longitudinally at baseline, one and six months. FPD, diameter and position were measured and participants rated intensity perception of sucrose, caffeine, menthol and capsaicin solutions. Salivary flow rate, protein concentration and relative changes in protein composition were measured following each tastant. FPD, diameter and position were unchanged at six months. FPD did not correlate with intensity rating for any taste. FPD did correlate with changes in salivary protein output following sucrose (ρ = 0.72, p = 0.02) and changes in levels of proline-rich protein and mucin 7 following capsaicin (ρ = 0.71, p = 0.02, ρ = 0.68, p = 0.04, respectively). These results suggest that over six months fungiform papillae are anatomically stable, playing a greater role in mediating the physiological salivary response to stimuli rather than determining the perceived intensity of taste.

ITGA2 as a potential nanotherapeutic target for glioblastoma.

High grade gliomas, including glioblastoma (GBM), are the most common and deadly brain cancers in adults. Here, we performed a quantitative and unbiased screening of 70 cancer-related antigens using comparative flow cytometry and, for the first time, identified integrin alpha-2 (ITGA2) as a novel molecular target for GBM. In comparison to epidermal growth factor receptor (EGFR), a well-established GBM target, ITGA2 is significantly more expressed on human GBM cells and significantly less expressed on normal human glial cells. We also found that ITGA2 antibody blockade significantly impedes GBM cell migration but not GBM cell proliferation. To investigate the utility of ITGA2 as a therapeutic target in GBM, we designed and engineered an ITGA2 antibody-directed liposome that can selectively deliver doxorubicin, a standard-of-care chemotherapeutic agent, to GBM cells. This novel approach significantly improved antitumor efficacy. We also demonstrated that these ITGA2 antibody-directed liposomes can effectively breach the blood-brain tumor barrier (BBTB) in vitro via GBM-induced angiogenesis effects. These findings support further research into the use of ITGA2 as a novel nanotherapeutic target for GBM.

Determining bacterial and host contributions to the human salivary metabolome.

BACKGROUND: Salivary metabolomics is rapidly advancing. AIM AND METHODS: To determine the extent to which salivary metabolites reflects host or microbial metabolic activity whole-mouth saliva (WMS), parotid saliva (PS) and plasma collected contemporaneously from healthy volunteers were analysed by 1H-NMR spectroscopy. Spectra underwent principal component analysis and k-means cluster analysis and metabolite quantification. WMS samples were cultured on both sucrose and peptide-enriched media. Correlation between metabolite concentration and bacterial load was assessed. RESULTS: WMS contained abundant short-chain fatty acids (SCFAs), which were minimal in PS and plasma. WMS spectral exhibited greater inter-individual variation than those of PS or plasma (6.7 and 3.6 fold, respectively), likely reflecting diversity of microbial metabolomes. WMS bacterial load correlated strongly with SCFA levels. Additional WMS metabolites including amines, amino acids and organic acids were positively correlated with bacterial load. Lactate, urea and citrate appeared to enter WMS via PS and the circulation. Urea correlated inversely with WMS bacterial load. CONCLUSIONS: Oral microbiota contribute significantly to the WMS metabolome. Several WMS metabolites (lactate, urea and citrate) are derived from the host circulation. WMS may be particularly useful to aid diagnosis of conditions reflective of dysbiosis. WMS could also complement other gastrointestinal fluids in future metabolomic studies.

Light Chain Diversity among the Botulinum Neurotoxins.

Botulinum neurotoxins (BoNT) are produced by several species of clostridium. There are seven immunologically unique BoNT serotypes (A⁻G). The Centers for Disease Control classifies BoNTs as 'Category A' select agents and are the most lethal protein toxins for humans. Recently, BoNT-like proteins have also been identified in several non-clostridia. BoNTs are di-chain proteins comprised of an N-terminal zinc metalloprotease Light Chain (LC) and a C-terminal Heavy Chain (HC) which includes the translocation and receptor binding domains. The two chains are held together by a disulfide bond. The LC cleaves Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The cleavage of SNAREs inhibits the fusion of synaptic vesicles to the cell membrane and the subsequent release of acetylcholine, which results in flaccid paralysis. The LC controls the catalytic properties and the duration of BoNT action. This review discusses the mechanism for LC catalysis, LC translocation, and the basis for the duration of LC action. Understanding these properties of the LC may expand the applications of BoNT as human therapies.