Visualizing the chaperone-mediated folding trajectory of the G protein ß5 ß-propeller.

The Chaperonin Containing Tailless polypeptide 1 (CCT) complex is an essential protein folding machine with a diverse clientele of substrates, including many proteins with ß-propeller domains. Here, we determine the structures of human CCT in complex with its accessory co-chaperone, phosducin-like protein 1 (PhLP1), in the process of folding Gß5, a component of Regulator of G protein Signaling (RGS) complexes. Cryoelectron microscopy (cryo-EM) and image processing reveal an ensemble of distinct snapshots that represent the folding trajectory of Gß5 from an unfolded molten globule to a fully folded ß-propeller. These structures reveal the mechanism by which CCT directs Gß5 folding through initiating specific intermolecular contacts that facilitate the sequential folding of individual ß sheets until the propeller closes into its native structure. This work directly visualizes chaperone-mediated protein folding and establishes that CCT orchestrates folding by stabilizing intermediates through interactions with surface residues that permit the hydrophobic core to coalesce into its folded state.

Penalized smoothing splines resolve the curvature identifiability problem in age-period-cohort models with unequal intervals.

Age-period-cohort (APC) models are frequently used in a variety of health and demographic-related outcomes. Fitting and interpreting APC models to data in equal intervals (equal age and period widths) is nontrivial due to the structural link between the three temporal effects (given two, the third can always be found) causing the well-known identification problem. The usual method for resolving the structural link identification problem is to base a model on identifiable quantities. It is common to find health and demographic data in unequal intervals, this creates further identification problems on top of the structural link. We highlight the new issues by showing that curvatures which were identifiable for equal intervals are no longer identifiable for unequal data. Furthermore, through extensive simulation studies, we show how previous methods for unequal APC models are not always appropriate due to their sensitivity to the choice of functions used to approximate the true temporal functions. We propose a new method for modeling unequal APC data using penalized smoothing splines. Our proposal effectively resolves the curvature identification issue that arises and is robust to the choice of the approximating function. To demonstrate the effectiveness of our proposal, we conclude with an application to UK all-cause mortality data from the Human mortality database.

Promoting health equity with HBCUs: Breaking away from structural racism.

BACKGROUND: The United States continues to be plagued with pervasive health disparities. Leading health and professional organizations acknowledge structural racism as a contributing factor for the lack of a racially diverse nursing workforce particularly those serving in leadership roles which could help to mitigate health disparities among historically stigmatized populations. PURPOSE: Purpose Lack of funding for Historically Black Colleges and Universities (HBCUs) and lack of meaningful partnerships, stymie efforts that can be made by nursing programs at HBCUs. DISCUSSION: Discussion This position paper examines collaborative actions that can address upstream factors that perpetuate healthcare disparities through deep engagement between the policymakers, professional associations, industry, and educational institutions. METHODS: Faculty representing HBCU's and predominately White institutions, professional organizations, and staff met via videoconference to refine the focus of the paper, determine topic areas for writing teams, and refine details which occurred during weekly meetings. CONCLUSION: To disengage from structural racism, three critical recommendations are amplified with associated examples.

Validity and Reliability of the Turkish Version of the Revised Low Vision Independence Measure (LVIM-R).

IMPORTANCE: Few tools are available for assessing the activity performance of people with low vision. Additional practical instruments are required for occupational therapy evaluation. OBJECTIVE: To evaluate the validity and reliability of the Turkish version of the Revised Low Vision Independence Measure (LVIM-R). DESIGN: We analyzed Turkish LVIM-R items using confirmatory factor analysis with parceling. Test-retest reliability was measured using the intraclass correlation coefficient (ICC). The study was conducted between April 2020 and July 2021. SETTING: Ophthalmology clinic. PARTICIPANTS: The study included 89 people with low vision who applied to an ophthalmology training and research clinic. Participants were age ≥18 yr, had visual acuity of 0.5 to 1.3 logarithms of the minimum angle of resolution, and consented to participate. INTERVENTION: Not applicable. OUTCOMES AND MEASURES: Turkish version of the LVIM-R, a self-report measure of activity performance for people with low vision. RESULTS: In the principal-components analysis, the highest eigenvalue was Armor's θ = .966. ICCs were .995 for Visual Field or Scotoma, .997 for Visual Acuity, and .997 for Total score. Other results included χ2 = 9.566 (p = .297), comparative fit index = .998, Tucker-Lewis Index = .996, and root mean square error of approximation = .047. Goodness-of-fit indices support the structural validity of the two-factor model. CONCLUSION: The Turkish version of the LVIM-R is a valid and reliable assessment tool for occupational therapy practice in low vision rehabilitation. What This Article Adds: The Turkish LVIM-R is a valid and reliable assessment for use in low vision rehabilitation.

Mechanistic insights into protein folding by the eukaryotic chaperonin complex CCT.

The cytosolic chaperonin CCT is indispensable to eukaryotic life, folding the cytoskeletal proteins actin and tubulin along with an estimated 10% of the remaining proteome. However, it also participates in human diseases such as cancer and viral infections, rendering it valuable as a potential therapeutic target. CCT consists of two stacked rings, each comprised of eight homologous but distinct subunits, that assists the folding of a remarkable substrate clientele that exhibits both broad diversity and specificity. Much of the work in recent years has been aimed at understanding the mechanisms of CCT substrate recognition and folding. These studies have revealed new binding sites and mechanisms by which CCT uses its distinctive subunit arrangement to fold structurally unrelated substrates. Here, we review recent structural insights into CCT-substrate interactions and place them into the broader context of CCT function and its implications for human health.

Using Bayesian networks to identify musculoskeletal symptoms influencing the risk of developing psoriatic arthritis in people with psoriasis.

OBJECTIVES: The aim of this study was to explore the use of Bayesian networks (BNs) to understand the relationships between musculoskeletal symptoms and the development of PsA in people with psoriasis. METHODS: Incident cases of psoriasis were identified for 1998 to 2015 from the UK Clinical Research Practice Datalink. Musculoskeletal symptoms (identified by Medcodes) were concatenated into primary groups, each made up of several subgroups. Baseline demographics for gender, age, BMI, psoriasis severity, alcohol use and smoking status were also extracted. Several BN structures were composed using a combination of expert knowledge and data-oriented modelling based on: (i) primary musculoskeletal symptom groups; (ii) musculoskeletal symptom subgroups and (iii) demographic variables. Predictive ability of the networks using the area under the receiver operating characteristic curve was calculated. RESULTS: Over one million musculoskeletal symptoms were extracted for the 90 189 incident cases of psoriasis identified, of which 1409 developed PsA. The BN analysis yielded direct relationships between gender, BMI, arthralgia, finger pain, fatigue, hand pain, hip pain, knee pain, swelling, back pain, myalgia and PsA. The best BN, achieved by using the more site-specific musculoskeletal symptom subgroups, was 76% accurate in predicting the development of PsA in a test set and had an area under the receiver operating characteristic curve of 0.73 (95% CI: 0.70, 0.75). CONCLUSION: The presented BN model may be a useful method to identify clusters of symptoms that predict the development of PsA with reasonable accuracy. Using a BN approach, we have shown that there are several symptoms which are predecessors of PsA, including fatigue, specific types of pain and swelling.

Challenges in realising the potential of wastewater-based epidemiology to quantitatively monitor and predict the spread of disease.

Researchers around the world have demonstrated correlations between measurements of SARS-CoV-2 RNA in wastewater (WW) and case rates of COVID-19 derived from direct testing of individuals. This has raised concerns that wastewater-based epidemiology (WBE) methods might be used to quantify the spread of this and other diseases, perhaps faster than direct testing, and with less expense and intrusion. We illustrate, using data from Scotland and the USA, the issues regarding the construction of effective predictive models for disease case rates. We discuss the effects of variation in, and the problem of aligning, public health (PH) reporting and WW measurements. We investigate time-varying effects in PH-reported case rates and their relationship to WW measurements. We show the lack of proportionality of WW measurements to case rates with associated spatial heterogeneity. We illustrate how the precision of predictions is affected by the level of aggregation chosen. We determine whether PH or WW measurements are the leading indicators of disease and how they may be used in conjunction to produce predictive models. The prospects of using WW-based predictive models with or without ongoing PH data are discussed.

Isisekelo Sempilo study protocol for the effectiveness of HIV prevention embedded in sexual health with or without peer navigator support (Thetha Nami) to reduce prevalence of transmissible HIV amongst adolescents and young adults in rural KwaZulu-Natal: a 2 × 2 factorial randomised controlled trial.

BACKGROUND: Antiretroviral therapy (ART) through universal test and treat (UTT) and HIV pre-exposure prophylaxis (PrEP) substantially reduces HIV-related mortality, morbidity and incidence. Effective individual-level prevention modalities have not translated into population-level impact in southern Africa due to sub-optimal coverage among adolescents and youth who are hard to engage. We aim to investigate the feasibility, acceptability, and preliminary population level effectiveness of HIV prevention services with or without peer support to reduce prevalence of transmissible HIV amongst adolescents and young adults in KwaZulu-Natal. METHODS: We are conducting a 2 × 2 factorial trial among young men and women aged 16-29 years, randomly selected from the Africa Health Research Institute demographic surveillance area. Participants are randomly allocated to one of four intervention combinations: 1) Standard of Care (SOC): nurse-led services for HIV testing plus ART if positive or PrEP for those eligible and negative; 2) Sexual and Reproductive Health (SRH): Baseline self-collected vaginal and urine samples with study-organized clinic appointments for results, treatment and delivery of HIV testing, ART and PrEP integrated with SRH services; 3) Peer-support: Study referral of participants to a peer navigator to assess their health, social and educational needs and provide risk-informed HIV prevention, including facilitating clinic attendance; or 4) SRH + peer-support. The primary outcomes for effectiveness are: (1) the proportion of individuals with infectious HIV at 12 months and (2) uptake of risk-informed comprehensive HIV prevention services within 60 days of enrolment. At 12 months, all participants will be contacted at home and the study team will collect a dried blood spot for HIV ELISA and HIV viral load testing. DISCUSSION: This trial will enable us to understand the relative importance of SRH and peer support in creating demand for effective and risk informed biomedical HIV prevention and preliminary data on their effectiveness on reducing the prevalence of transmissible HIV amongst all adolescents and youth. TRIAL REGISTRATION: Trial Registry: clincialtrials.gov. CLINICALTRIALS: gov Identifier NCT04532307 . Registered: March 2020.

The roles of intolerance of uncertainty, anxiety sensitivity and distress tolerance in hoarding disorder compared with OCD and healthy controls.

BACKGROUND: It is suggested that the different psychological vulnerability factors of intolerance of uncertainty (IU), anxiety sensitivity (AS) and distress tolerance (DT) may be in important in hoarding disorder (HD). However, the extent to which these factors are specific to HD compared with other disorders remains unclear. AIMS: The current study aimed to investigate differences in IU, AS and DT in three groups: HD (n=66), obsessive compulsive disorder (OCD; n=59) and healthy controls (HCs; n=63). METHOD: Participants completed an online battery of standardised self-report measures to establish the independent variable of group membership (HD, OCD and HC) and the dependent variables (IU, AS and DT). RESULTS: A MANOVA analysis indicated statistically significant differences in IU, AS and DT between the clinical groups and HCs. Follow-up analyses showed no statistically significant differences between the HD and OCD group for any of the three constructs. The results remained the same when examining the effects of co-morbid HD and OCD. An unexpected finding was the trend for IU, AS and DT to be more severe when HD and OCD were co-morbid. CONCLUSIONS: The evidence suggests the absence of a specific relationship between IU, AS or DT in HD and instead is consistent with existing research which suggests that these psychological vulnerability factors are transdiagnostic constructs across anxiety disorders. The implications of the findings are discussed.

Smooth muscle tumors of the gastrointestinal tract: an analysis of prognostic features in 407 cases.

Smooth muscle tumors represent the second most common mural mesenchymal neoplasm in the gastrointestinal tract, but established criteria for prognostic assessment of these tumors are lacking. A large cohort of surgically resected intramural gastrointestinal smooth muscle tumors from 31 institutions was analyzed to identify potential prognostic features. Pathologic features were assessed by expert gastrointestinal and/or soft tissue pathologists at each center. Immunohistochemical confirmation was required. A total of 407 cases from the esophagus (n = 97, 24%), stomach (n = 180, 44%), small bowel (n = 74, 18%), and colorectum (n = 56, 14%) were identified. Patients ranged in age from 19 to 92 years (mean 55 years), with a slight female predominance (57%). Mean tumor size was 5.4 cm, with the largest tumor measuring 29 cm. Disease progression following surgery, defined as local recurrence, metastasis, or disease-related death, occurred in 56 patients (14%). Colorectal tumors were most likely to progress, followed by small bowel and gastric tumors. None of the esophageal tumors in this series progressed. Receiver operator characteristic analysis identified optimal cutoffs of 9.8 cm and 3 mitoses/5 mm2 for discriminating between progressive and non-progressive tumors. Histologic features strongly associated with progression by univariate analysis included moderate-to-severe atypia, high cellularity, abnormal differentiation (defined as differentiation not closely resembling that of normal smooth muscle), tumor necrosis, mucosal ulceration, lamina propria involvement, and serosal involvement (P < 0.0001 for all features). Age, sex, and margin status were not significantly associated with progression (P = 0.23, 0.82, and 0.07, respectively). A risk assessment table was created based on tumor site, size, and mitotic count, and Kaplan-Meier plots of progression-free survival for each subgroup revealed progression-based tiers. Based on our findings, it appears that nonesophageal gastrointestinal smooth muscle tumors measuring >10 cm and/or showing ≥3 mitoses/5 mm2 may behave aggressively, and therefore close clinical follow-up is recommended in these cases.

Responsiveness of the Revised Low Vision Independence Measure (LVIM-R).

IMPORTANCE: The Low Vision Independence Measure (LVIM) was designed to measure the effectiveness of occupational therapy to improve visual ability in low vision rehabilitation. OBJECTIVE: To validate the Revised LVIM (LVIM-R) as an outcome measure by determining its sensitivity to visual ability changes after occupational therapy. DESIGN: In this observational study, LVIM-R scores were collected before and after customary low vision intervention. SETTING: Home health agency or outpatient facility. PARTICIPANTS: Forty-four participants with a mean age of 80.2 yr (standard deviation = 11.2) and an average length of visual impairment of 6.2 yr. Twenty-three participants (52.3%) were treated in an outpatient setting, and 21 (47.7%) were treated in the home. The majority of participants were non-Hispanic White (92.8%) and had macular degeneration (54.5%). INTERVENTION: Customary low vision intervention by occupational therapists. OUTCOMES AND MEASURES: LVIM-R scores were collected before and after intervention, and participants' pre- and posttest scores were anchored into Rasch-calibrated item parameters. RESULTS: Paired t tests demonstrated significant increases in person measures with large effect sizes for both constructs of the LVIM-R, the visual field or scotoma (t[43] = 6.46, p < .0001; Cohen's d = 0.92) and visual acuity (t[43] = 9.08, p < .0001; Cohen's d = 1.14) constructs. CONCLUSIONS AND RELEVANCE: The LVIM-R is sensitive to changes in visual ability in clients who have good rehabilitation potential and can be used to examine the effectiveness of occupational therapy for low vision. WHAT THIS ARTICLE ADDS: The LVIM-R is useful for measuring occupational therapy outcomes in low vision rehabilitation in the home or in outpatient settings.

Measurement Validity of the Low Vision Independence Measure (LVIM).

OBJECTIVE: This study's objective was to test the psychometrics of Smith's (2013) Low Vision Independence Measure (LVIM) using the Rasch model. METHOD: A cohort design was used with 93 participants receiving occupational therapy for low vision. Confirmatory factor analysis (CFA) with a parceling approach was used to test the LVIM factors and Rasch analysis to examine item-level psychometrics. RESULTS: Participants' average age was 78.9 yr (standard deviation = 12.1), and the majority were female (72.8%) with macular degeneration (62.3%). The CFA revealed two measurement factors: visual field or scotoma (n = 28) and visual acuity (n = 24). We removed six misfitting items, and the two factors of the revised LVIM demonstrated good rating scale function, good internal consistency (person reliability: visual field, .87; visual acuity, .90), good precision (person strata: visual field, 3.91; visual acuity, 4.40), no ceiling or floor effects, and no differential item functioning. CONCLUSION: The revised LVIM demonstrates good psychometrics on the Rasch model and can be used as a valid outcome measure in low vision rehabilitation.

Differentiating Botulinum Neurotoxin-Producing Clostridia with a Simple, Multiplex PCR Assay.

Diverse members of the genus Clostridium produce botulinum neurotoxins (BoNTs), which cause a flaccid paralysis known as botulism. While multiple species of clostridia produce BoNTs, the majority of human botulism cases have been attributed to Clostridium botulinum groups I and II. Recent comparative genomic studies have demonstrated the genomic diversity within these BoNT-producing species. This report introduces a multiplex PCR assay for differentiating members of C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Coding region sequences unique to each of the four species/subgroups were identified by in silico analyses of thousands of genome assemblies, and PCR primers were designed to amplify each marker. The resulting multiplex PCR assay correctly assigned 41 tested isolates to the appropriate species or subgroup. A separate PCR assay to determine the presence of the ntnh gene (a gene associated with the botulinum neurotoxin gene cluster) was developed and validated. The ntnh gene PCR assay provides information about the presence or absence of the botulinum neurotoxin gene cluster and the type of gene cluster present (ha positive [ha+] or orfX+). The increased availability of whole-genome sequence data and comparative genomic tools enabled the design of these assays, which provide valuable information for characterizing BoNT-producing clostridia. The PCR assays are rapid, inexpensive tests that can be applied to a variety of sample types to assign isolates to species/subgroups and to detect clostridia with botulinum neurotoxin gene (bont) clusters.IMPORTANCE Diverse clostridia produce the botulinum neurotoxin, one of the most potent known neurotoxins. In this study, a multiplex PCR assay was developed to differentiate clostridia that are most commonly isolated in connection with human botulism cases: C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Since BoNT-producing and nontoxigenic isolates can be found in each species, a PCR assay to determine the presence of the ntnh gene, which is a universally present component of bont gene clusters, and to provide information about the type (ha+ or orfX+) of bont gene cluster present in a sample was also developed. The PCR assays provide simple, rapid, and inexpensive tools for screening uncharacterized isolates from clinical or environmental samples. The information provided by these assays can inform epidemiological studies, aid with identifying mixtures of isolates and unknown isolates in culture collections, and confirm the presence of bacteria of interest.

Comparative genomic analyses reveal broad diversity in botulinum-toxin-producing Clostridia.

BACKGROUND: Clostridium botulinum is a diverse group of bacteria characterized by the production of botulinum neurotoxin. Botulinum neurotoxins are classified into serotypes (BoNT/A-G), which are produced by six species/Groups of Clostridia, but the genetic background of the bacteria remains poorly understood. The purpose of this study was to use comparative genomics to provide insights into the genetic diversity and evolutionary history of bacteria that produce the potent botulinum neurotoxin. RESULTS: Comparative genomic analyses of over 170 Clostridia genomes, including our draft genome assemblies for 59 newly sequenced Clostridia strains from six continents and publicly available genomic data, provided in-depth insights into the diversity and distribution of BoNT-producing bacteria. These newly sequenced strains included Group I and II strains that express BoNT/A,/B,/E, or/F as well as bivalent strains. BoNT-producing Clostridia and closely related Clostridia species were delineated with a variety of methods including 16S rRNA gene, concatenated marker genes, core genome and concatenated multi-locus sequencing typing (MLST) gene phylogenies that related whole genome sequenced strains to publicly available strains and sequence types. These analyses illustrated the phylogenetic diversity in each Group and the diversity of genomic backgrounds that express the same toxin type or subtype. Comparisons of the botulinum neurotoxin genes did not identify novel toxin types or variants. CONCLUSIONS: This study represents one of the most comprehensive analyses of whole genome sequence data for Group I and II BoNT-producing strains. Read data and draft genome assemblies generated for 59 isolates will be a resource to the research community. Core genome phylogenies proved to be a powerful tool for differentiating BoNT-producing strains and can provide a framework for the study of these bacteria. Comparative genomic analyses of Clostridia species illustrate the diversity of botulinum-neurotoxin-producing strains and the plasticity of the genomic backgrounds in which bont genes are found.

Improved Cardiorespiratory Fitness Is Associated with Increased Cortical Thickness in Mild Cognitive Impairment.

Cortical atrophy is a biomarker of Alzheimer's disease (AD) that correlates with clinical symptoms. This study examined changes in cortical thickness from before to after an exercise intervention in mild cognitive impairment (MCI) and healthy elders. Thirty physically inactive older adults (14 MCI, 16 healthy controls) underwent MRI before and after participating in a 12-week moderate intensity walking intervention. Participants were between the ages of 61 and 88. Change in cardiorespiratory fitness was assessed using residualized scores of the peak rate of oxygen consumption (V̇O2peak) from pre- to post-intervention. Structural magnetic resonance images were processed using FreeSurfer v5.1.0. V̇O2peak increased an average of 8.49%, which was comparable between MCI and healthy elders. Overall, cortical thickness was stable except for a significant decrease in the right fusiform gyrus in both groups. However, improvement in cardiorespiratory fitness due to the intervention (V̇O2peak) was positively correlated with cortical thickness change in the bilateral insula, precentral gyri, precuneus, posterior cingulate, and inferior and superior frontal cortices. Moreover, MCI participants exhibited stronger positive correlations compared to healthy elders in the left insula and superior temporal gyrus. A 12-week moderate intensity walking intervention led to significantly improved fitness in both MCI and healthy elders. Improved V̇O2peak was associated with widespread increased cortical thickness, which was similar between MCI and healthy elders. Thus, regular exercise may be an especially beneficial intervention to counteract cortical atrophy in all risk groups, and may provide protection against future cognitive decline in both healthy elders and MCI.

Determinants of butyrylcholinesterase inhibition among agricultural pesticide handlers in Washington State: an update.

OBJECTIVES: Organophosphate (OP) and N-methyl-carbamate (CB) insecticides are used widely in agriculture to manage insect pests of economic importance. Agricultural workers are more likely to suffer exposure because of the widespread use of OP/CBs in agriculture, and pesticide-related illnesses among handlers may be more severe when compared to other farm workers. The goal of this study was to identify occupational and personal characteristics associated with butyrylcholinesterase (BuChE) inhibition in participants recruited from the Washington State Cholinesterase Monitoring Program from 2006 to 2011. METHODS: We conducted a longitudinal study among agricultural pesticide handlers in Washington State during the OP/CB spray season (March-July) over a 6-year period (2006-2011). Linear mixed effects regression models were used to evaluate BuChE inhibition in relation to self-reported occupational and personal characteristics. RESULTS: Relative to pre-season baseline levels, the mean decrease in BuChE activity during the OP/CB spray season over all years of the study period was 3.77% (P < 0.001). Greater BuChE inhibition was observed among handlers who reported using multiple OP/CBs (ß = -2.70, P = 0.045), mixed or loaded OP/CBs (ß = -3.97, P = 0.002), did not store personal protective equipment (PPE) in a locker at work (ß = -3.4, P = 0.014), or did not wear chemical-resistant boots (ß = -16.6, P < 0.001). DISCUSSION AND CONCLUSIONS: The Washington State Cholinesterase Monitoring Program has provided a valuable opportunity to evaluate potential sources of OP/CB exposure among agricultural pesticide handlers. Several previously reported associations were confirmed in the current analysis, which included a larger number of pesticide handlers enrolled over a longer time period. The use of multiple OP/CBs and mixing/loading activities were significant risk factors, and the use of chemical-resistant boots and lockers for PPE storage were protective factors. Our findings point toward logical interventions to reduce exposure such as the implementation of engineering controls for mixing/loading activities, requirements for appropriate footwear, and the regular use of lockers for PPE storage.

Centers for disease control and prevention expert panel meetings on prevention and treatment of anthrax in adults.

The Centers for Disease Control and Prevention convened panels of anthrax experts to review and update guidelines for anthrax postexposure prophylaxis and treatment. The panels included civilian and military anthrax experts and clinicians with experience treating anthrax patients. Specialties represented included internal medicine, pediatrics, obstetrics, infectious disease, emergency medicine, critical care, pulmonology, hematology, and nephrology. Panelists discussed recent patients with systemic anthrax; reviews of published, unpublished, and proprietary data regarding antimicrobial drugs and anthrax antitoxins; and critical care measures of potential benefit to patients with anthrax. This article updates antimicrobial postexposure prophylaxis and antimicrobial and antitoxin treatment options and describes potentially beneficial critical care measures for persons with anthrax, including clinical procedures for infected nonpregnant adults. Changes from previous guidelines include an expanded discussion of critical care and clinical procedures and additional antimicrobial choices, including preferred antimicrobial drug treatment for possible anthrax meningitis.

Three enzymatically active neurotoxins of Clostridium botulinum strain Af84: BoNT/A2, /F4, and /F5.

Botulinum neurotoxins (BoNTs) are produced by various species of clostridia and are potent neurotoxins which cause the disease botulism, by cleaving proteins needed for successful nerve transmission. There are currently seven confirmed serotypes of BoNTs, labeled A-G, and toxin-producing clostridia typically only produce one serotype of BoNT. There are a few strains (bivalent strains) which are known to produce more than one serotype of BoNT, producing either both BoNT/A and /B, BoNT/A and /F, or BoNT/B and /F, designated as Ab, Ba, Af, or Bf. Recently, it was reported that Clostridium botulinum strain Af84 has three neurotoxin gene clusters: bont/A2, bont/F4, and bont/F5. This was the first report of a clostridial organism containing more than two neurotoxin gene clusters. Using a mass spectrometry based proteomics approach, we report here that all three neurotoxins, BoNT/A2, /F4, and /F5, are produced by C. botulinum Af84. Label free MS(E) quantification of the three toxins indicated that toxin composition is 88% BoNT/A2, 1% BoNT/F4, and 11% BoNT/F5. The enzymatic activity of all three neurotoxins was assessed by examining the enzymatic activity of the neurotoxins upon peptide substrates, which mimic the toxins' natural targets, and monitoring cleavage of the substrates by mass spectrometry. We determined that all three neurotoxins are enzymatically active. This is the first report of three enzymatically active neurotoxins produced in a single strain of Clostridium botulinum.

A two-stage multiplex method for quantitative analysis of botulinum neurotoxins type A, B, E, and F by MALDI-TOF mass spectrometry.

In this publication, we report on the development of a quantitative enzymatic method for the detection of four botulinum neurotoxin (BoNT) serotypes responsible for human botulism by MALDI-TOF mass spectrometry. Factors that might affect the linearity and dynamic range for detection of BoNT cleavage products were initially examined, including the amount of peptide substrate and internal standard, the timing of cleavage reaction, and the components in the reaction solution. It was found that a long incubation time produced sensitive results, but was not capable of determining higher toxin concentrations, whereas a short incubation time was less sensitive so that lower toxin concentrations were not detected. In order to overcome these limitations, a two-stage analysis strategy was applied. The first stage analysis involved a short incubation period (e.g., 30 min). If no toxin was detected at this stage, the cleavage reaction was allowed to continue and the samples were analyzed at a second time point (4 h), so that toxin levels lower than 1 mouse LD50 or 55 attomoles per milliliter (55 amol/mL) could be quantified. By combining the results from two-stage quantification, 4 or 5 orders of magnitude in dynamic range were achieved for the detection of the serotypes of BoNT/A, BoNT/B, BoNT/E, or BoNT/F. The effect of multiplexing the assay by mixing substrates for different BoNT serotypes into a single reaction was also investigated in order to reduce the numbers of the cleavage reactions and save valuable clinical samples.

Genetic diversity within Clostridium botulinum serotypes, botulinum neurotoxin gene clusters and toxin subtypes.

Clostridium botulinum is a species of spore-forming anaerobic bacteria defined by the expression of any one or two of seven serologically distinct botulinum neurotoxins (BoNTs) designated BoNT/A-G. This Gram-positive bacterium was first identified in 1897 and since then the paralyzing and lethal effects of its toxin have resulted in the recognition of different forms of the intoxication known as food-borne, infant, or wound botulism. Early microbiological and biochemical characterization of C. botulinum isolates revealed that the bacteria within the species had different characteristics and expressed different toxin types. To organize the variable bacterial traits within the species, Group I-IV designations were created. Interestingly, it was observed that isolates within different Groups could express the same toxin type and conversely a single Group could express different toxin types. This discordant phylogeny between the toxin and the host bacteria indicated that horizontal gene transfer of the toxin was responsible for the variation observed within the species. The recent availability of multiple C. botulinum genomic sequences has offered the ability to bioinformatically analyze the locations of the bont genes, the composition of their toxin gene clusters, and the genes flanking these regions to understand their variation. Comparison of the genomic sequences representing multiple serotypes indicates that the bont genes are not in random locations. Instead the analyses revealed specific regions where the toxin genes occur within the genomes representing serotype A, B, C, E, and F C. botulinum strains and C. butyricum type E strains. The genomic analyses have provided evidence of horizontal gene transfer, site-specific insertion, and recombination events. These events have contributed to the variation observed among the neurotoxins, the toxin gene clusters and the bacteria that contain them, and has supported the historical microbiological, and biochemical characterization of the Group classification within the species.