Neutralization of ricin toxin on building interior surfaces using liquid decontaminants.

Ricin is a highly toxic protein, capable of inhibiting protein synthesis within cells, and is produced from the beans of the Ricinus communis (castor bean) plant. Numerous recent incidents involving ricin have occurred, many in the form of mailed letters resulting in both building and mail sorting facility contamination. The goal of this study was to assess the decontamination efficacy of several commercial off-the-shelf (COTS) cleaners and decontaminants (solutions of sodium hypochlorite [bleach], quaternary ammonium, sodium percarbonate, peracetic acid, and hydrogen peroxide) against a crude preparation of ricin toxin. The ricin was inoculated onto four common building materials (pine wood, drywall joint tape, countertop laminate, and industrial carpet), and the decontaminants were applied to the test coupons using a handheld sprayer. Decontamination efficacy was quantified using an in-vitro cytotoxicity assay to measure the quantity of bioactive ricin toxin extracted from test coupons as compared to the corresponding positive controls (not sprayed with decontaminant). Results showed that decontamination efficacy varied by decontaminant and substrate material, and that efficacy generally improved as the number of spray applications or contact time increased. The solutions of 0.45% peracetic acid and the 20,000-parts per million (ppm) sodium hypochlorite provided the overall best decontamination efficacy. The 0.45% peracetic acid solution achieved 97.8 to 99.8% reduction with a 30-min contact time.

Changing structures, changing paradigms: NELF helps regulate paused or elongating RNA polymerase II.

Using cryo-EM and biochemical methods, Su and Vos1 discover an alternative NELF structural state that enables transcription and switches NELF-RNA polymerase II (RNAPII) compatibility with other RNAPII-associated factors that regulate pausing, elongation, termination, and transcription-coupled DNA repair.

Evaluation of altered environmental conditions as a decontamination approach for SARS-CoV-2 when applied to aircraft related materials.

AIMS: The purpose of this study was to evaluate the effects of altered environmental conditions, specifically elevated temperature at various levels of expected relative humidity (RH), on the inactivation of SARS-CoV-2 when applied to U.S. Air Force aircraft materials. METHODS AND RESULTS: SARS CoV-2 (USA-WA1/2020) was spiked (∼1 × 105 TCID50) in either synthetic saliva or lung fluid, dried onto porous (e.g. Nylon strap) and nonporous materials (e.g. bare aluminum, silicone, and ABS plastic), placed in a test chamber and exposed to environmental conditions ranging from 40 to 51.7 °C and RH ranging from 0% to 50%. The amount of infectious SARS-CoV-2 was then assessed at various timepoints from 0 to 2 days. Warmer test temperatures, higher RH, and longer exposure duration resulted in higher inactivation rates per material type. Synthetic saliva inoculation vehicle was more readily decontaminated compared to materials inoculated with synthetic lung fluid. CONCLUSIONS: SARS-CoV-2 was readily inactivated below limit of quantitation (LOQ) for all materials inoculated using synthetic saliva vehicle within 6 hours when exposed to environmental conditions of 51.7 °C and RH ≥ 25%. Synthetic lung fluid vehicle did not follow the general trend of an increase in RH resulting in increased efficacy. The lung fluid performed best at the 20%-25% RH range to achieve complete inactivation below LOQ.

Evaluation of steam heat as a decontamination approach for SARS-CoV-2 when applied to common transit-related materials.

AIMS: The purpose of this study was to evaluate the efficacy of steam heat for inactivation of SARS-CoV-2 when applied to materials common in mass transit installations. METHODS AND RESULTS: SARS CoV-2 (USA-WA1/2020) was resuspended in either cell culture media or synthetic saliva, inoculated (∼1 × 106 TCID50) onto porous and nonporous materials and subjected to steam inactivation efficacy tests as either wet or dried droplets. The inoculated test materials were exposed to steam heat ranging from 70°C to 90°C. The amount of infectious SARS-CoV-2 remaining after various exposure durations ranging from 1 to 60 s was assessed. Higher steam heat application resulted in higher inactivation rates at short contact times. Steam applied at 1-inch distance (∼90°C at the surface) resulted in complete inactivation for dry inoculum within 2 s of exposure (excluding two outliers of 19 test samples at the 5-s duration) and within 2-30 s of exposure for wet droplets. Increasing the distance to 2 inches (∼70°C) also increased the exposure time required to achieve complete inactivation to 15 or 30 s for materials inoculated with saliva or cell culture media, respectively. CONCLUSIONS: Steam heat can provide high levels of decontamination (>3 log reduction) for transit-related materials contaminated with SARS-CoV-2 using a commercially available steam generator with a manageable exposure time of 2-5 s.

Passenger vehicle interior decontamination by low concentration hydrogen peroxide vapor following a wide area biological contamination incident.

AIMS: To assess low concentration hydrogen peroxide (LCHP) (H2O2) vapor dispersed with a home humidifier for its ability to decontaminate vehicle interiors contaminated with Bacillus anthracis surrogate Bacillus atrophaeus spores. METHODS AND RESULTS: Efficacy of a vaporized 3% H2O2 solution was evaluated for liquid volumes, on/off vehicle heating, ventilation, and air conditioning (HVAC) system operations, and temperatures that ranged from 5 to 27°C. Survival of the spores was assessed by quantification of remaining viable spores with efficacy quantified in terms of mean log10 reduction. Decontamination efficacy after the 6-day dwell time increased when the 3% H2O2 liquid volume was doubled, increasing from 4-of-10 to 10-of-10 nondetects (zero colonies counted using standard dilution and filter plating) inside the vehicle cabin. Recirculating cabin air through the HVAC system during decontamination decreased efficacy to 6-of-10 non-detects. While no 6-log10 reduction in viable spores was observed on the cabin filter with the cabin filter kept in place, a 6-log10 reduction was achieved after its removal and placement in the cabin during treatment. CONCLUSIONS: Results from this study allow for informed decisions on the use of LCHP vapor as an effective decontamination approach for vehicle interiors.

Effectiveness of formaldehyde in various soil types as a wide area decontamination approach for Bacillus anthracis spores.

The purpose of this study was to evaluate and compare the decontamination efficacy of liquid formaldehyde solutions for three soil types (sand, loam, and clay) against spores of Bacillus anthracis (B.a.) and Bacillus atrophaeus. Approximately 1 x 108 colony forming units were inoculated into each sample. Through a series of six bench-scale experiments, two concentrations and two volumes of liquid formaldehyde solution were then added to the soil samples and allowed to remain in contact for either 24 or 48 hours. Decontamination efficacy was assessed at either 22° or 10°C with or without lids atop the sample jars. Complete inactivation (no spores recovered from the soil samples, typically providing > 7 log reduction) of B.a. occurred in all soil types in five of the six tests, while complete inactivation of B. atrophaeus was achieved in all soil types for three of the six tests. The results demonstrated a higher probability of complete inactivation of spores for samples that were covered, samples that received the higher volume of formaldehyde, and those contaminated with B.a. Overall, the use of liquid formaldehyde solution (2.5-5%) was highly effective in inactivating entire spore populations (typically > 107 CFU) for both B.a. and B. atrophaeus in the soil matrices studied. Covering the soil after application would allow for less formaldehyde solution to be used without impacting the overall efficacy of the process. The data from this study may aid in the selection of appropriate decontamination parameters when using liquid formaldehyde for soil remediation. The data may also aid in the decision to use B. atrophaeus as a surrogate for B.a. when performing further decontamination studies using liquid formalin solutions.

Decontamination efficacy of common liquid disinfectants against non-spore-forming biological agents in soil matrices.

AIMS: The purpose of this study was to evaluate decontamination efficacy, within three soil types, against Yersinia pestis, Burkholderia pseudomallei, and the Venezuelan Equine Encephalitis virus (VEEV). METHODS AND RESULTS: One of three liquid disinfectants (dilute bleach, Virkon-S or Klozur One) was added to three soil types (sand, loam, or clay) and allowed contact for four pre-spike durations: 0, 15, 30 and 60 min. Y. pestis, B. pseudomallei, or VEEV was then spiked into the soil (10 microliters or approx. 1 × 107  CFU or PFU into 1 g soil) and decontamination efficacy assessed at post-spike contact times of 10 or 60 min at ambient environmental conditions. Across all soil types, sandy soil resulted in the least quenching to all three disinfectants tested as shown by sustained decontamination efficacy across all pre-spike and post-spike timepoints. Clay and loam soil types exhibited quenching effects on the hypochlorite and peroxygen based disinfectants (dilute bleach and Virkon S) and in general resulted in decreased efficacy with increased pre-spike contact time. The sodium persulfate (Klozur One) performance was the most consistent across all soil types and pre-spike contact times, resulting in greater efficacy with increased post-spike time. CONCLUSIONS: Liquid disinfectants can provide high levels of decontamination in soil for both viral and non-spore-forming bacterial select agents. Hypochlorite and peroxygen based disinfectants used in soils containing higher organic content (loam or clay) may require extended contact times or re-application of liquid disinfectant, in as little as 15 min of application, to achieve a 6-log reduction. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide information for the performance of three disinfectants in soil against non-spore-forming select agents. These data may aid response decision makers following a biological contamination incident by informing the selection of disinfectant as well as the re-application time to achieve effective site remediation.

The Mediator complex as a master regulator of transcription by RNA polymerase II.

The Mediator complex, which in humans is 1.4 MDa in size and includes 26 subunits, controls many aspects of RNA polymerase II (Pol II) function. Apart from its size, a defining feature of Mediator is its intrinsic disorder and conformational flexibility, which contributes to its ability to undergo phase separation and to interact with a myriad of regulatory factors. In this Review, we discuss Mediator structure and function, with emphasis on recent cryogenic electron microscopy data of the 4.0-MDa transcription preinitiation complex. We further discuss how Mediator and sequence-specific DNA-binding transcription factors enable enhancer-dependent regulation of Pol II function at distal gene promoters, through the formation of molecular condensates (or transcription hubs) and chromatin loops. Mediator regulation of Pol II reinitiation is also discussed, in the context of transcription bursting. We propose a working model for Mediator function that combines experimental results and theoretical considerations related to enhancer-promoter interactions, which reconciles contradictory data regarding whether enhancer-promoter communication is direct or indirect. We conclude with a discussion of Mediator's potential as a therapeutic target and of future research directions.

Providing care to children from low and middle-income countries with complex surgical problems: An 18 year review.

PURPOSE: The burden of surgical disease in children from low and middle-income countries (LMICs) is becoming more recognized as significant and undertreated.  We recently reviewed our health system's experience with providing quaternary-level surgical care to children from LMICs through a partnership with World Pediatric Project (WPP), a not-for-profit organization. METHODS: A retrospective review was performed of all WPP-sponsored patients who received surgical care at our children's hospital from LMICs in the Caribbean and Central America from July 2000 to August 2018. RESULTS: Two hundred and fifty-five patients (average age: 5.9 ± 5.3 years; range: <1-18 years) from 14 countries received a total of 371 moderately to significantly complex operations from 10 pediatric surgical subspecialties, with cardiac, neurosurgery, craniofacial and general/thoracic surgical subspecialties being the most common. The average length of hospital stay was 10.7 ± 18.9 days.  All patients had the opportunity to follow-up with local providers and/or visiting WPP-sponsored surgical teams. 227 patients (93.8%) were seen by WPP providers or released to an in-country physician partnering with WPP. There were 21 (8.2%) total, minor and major, postoperative complications.  Five deaths (2.0%) occurred at our institution and 7 from disease progression, after returning to their home country. CONCLUSIONS: Providing complex surgical care to LMIC children in the US may help address a significant global burden.  This care can be provided by multiple subspecialists with excellent outcomes, good follow-up, and low complication and mortality rates.  Having a supportive health care system, volunteer surgeons, and an organization that manages logistics and provides financial support is essential. TYPE OF STUDY: Clinical research, retrospective review LEVEL OF EVIDENCE: Level IV.

Evaluation of environmental conditions as a decontamination approach for SARS-CoV-2 when applied to common library, archive and museum-related materials.

AIMS: The purpose of this study was to evaluate the effects of ambient or altered environmental conditions on the inactivation of SARS-CoV-2 applied to materials common in libraries, archives and museums. METHODS AND RESULTS: Porous and non-porous materials (e.g. paper, plastic protective book cover) were inoculated with approximately 1 × 105 TCID50 SARS CoV-2 (USA-WA1/2020), dried, placed within test chamber in either a stacked or unstacked configuration, and exposed to environmental conditions ranging from 4 to 29°C at 40 ± 10% relative humidity. The amount of infectious SARS-CoV-2 was then assessed at various timepoints from 0 to 10 days. Ambient conditions resulted in varying inactivation rates per material type. Virus inactivation rate decreased when materials were stacked or at colder temperatures. Virus inactivation rate increased when materials were unstacked or at warmer temperatures. CONCLUSIONS: SARS-CoV-2 at ambient conditions resulted in the inactivation of virus below limit of quantitation (LOQ) for all materials by Day 8. Warmer temperatures, for a subset of materials, increased SARS-CoV-2 inactivation, and all were

Non-canonical H3K79me2-dependent pathways promote the survival of MLL-rearranged leukemia.

MLL-rearranged leukemia depends on H3K79 methylation. Depletion of this transcriptionally activating mark by DOT1L deletion or high concentrations of the inhibitor pinometostat downregulates HOXA9 and MEIS1, and consequently reduces leukemia survival. Yet, some MLL-rearranged leukemias are inexplicably susceptible to low-dose pinometostat, far below concentrations that downregulate this canonical proliferation pathway. In this context, we define alternative proliferation pathways that more directly derive from H3K79me2 loss. By ICeChIP-seq, H3K79me2 is markedly depleted at pinometostat-downregulated and MLL-fusion targets, with paradoxical increases of H3K4me3 and loss of H3K27me3. Although downregulation of polycomb components accounts for some of the proliferation defect, transcriptional downregulation of FLT3 is the major pathway. Loss-of-FLT3-function recapitulates the cytotoxicity and gene expression consequences of low-dose pinometostat, whereas overexpression of constitutively active STAT5A, a target of FLT3-ITD-signaling, largely rescues these defects. This pathway also depends on MLL1, indicating combinations of DOT1L, MLL1 and FLT3 inhibitors should be explored for treating FLT3-mutant leukemia.

Formaldehyde Vapor Characteristics in Varied Decontamination Environments.

INTRODUCTION: This effort investigated formaldehyde vapor characteristics under various environmental conditions by the analyses of air samples collected over a time-course. This knowledge will help responders achieve desired formaldehyde exposure parameters for decontamination of affected spaces after a biological contamination incident. METHODS: Prescribed masses of paraformaldehyde and formalin were sublimated or evaporated, respectively, to generate formaldehyde vapor. Adsorbent cartridges were used to collect air samples from the test chamber at predetermined times. A validated method was used to extract the cartridges and analyze for formaldehyde via liquid chromatography. In addition, material demand for the formaldehyde was evaluated by inclusion of arrays of Plexiglas panels in the test chamber to determine the impact of varied surface areas within the test chamber. Temperature was controlled with a circulating water bath connected to a radiator and fan inside the chamber. Relative humidity was controlled with humidity fixed-point salt solutions and water vapor generated from evaporated water. RESULTS: Low temperature trials (approximately 10°C) resulted in decreased formaldehyde air concentrations throughout the 48-hour time-course when compared with formaldehyde concentrations in the ambient temperature trials (approximately 22°C). The addition of clear Plexiglas panels to increase the surface area of the test chamber interior resulted in appreciable decreases of formaldehyde air concentration when compared to an empty test chamber. CONCLUSION: This work has shown that environmental variables and surface-to-volume ratios in the decontaminated space may affect the availability of formaldehyde in the air and, therefore, may affect decontamination effectiveness.

Decontamination of Bacillus Spores with Formaldehyde Vapor under Varied Environmental Conditions.

Introduction: This study investigated formaldehyde decontamination efficacy against dried Bacillus spores on porous and non-porous test surfaces, under various environmental conditions. This knowledge will help responders determine effective formaldehyde exposure parameters to decontaminate affected spaces following a biological agent release. Methods: Prescribed masses of paraformaldehyde or formalin were sublimated or evaporated, respectively, to generate formaldehyde vapor within a bench-scale test chamber. Adsorbent cartridges were used to measure formaldehyde vapor concentrations in the chamber at pre-determined times. A validated method was used to extract the cartridges and analyze for formaldehyde via liquid chromatography. Spores of Bacillus globigii, Bacillus thuringiensis, and Bacillus anthracis were inoculated and dried onto porous bare pine wood and non-porous painted concrete material coupons. A series of tests was conducted where temperature, relative humidity, and formaldehyde concentration were varied, to determine treatment efficacy outside of conditions where this decontaminant is well-characterized (laboratory temperature and humidity and 12 mg/L theoretical formaldehyde vapor concentration) to predict decontamination efficacy in applications that may arise following a biological incident. Results: Low temperature trials (approximately 10°C) resulted in decreased formaldehyde air concentrations throughout the 48-hour time-course when compared with formaldehyde concentrations collected in the ambient temperature trials (approximately 22°C). Generally, decontamination efficacy on wood was lower for all three spore types compared with painted concrete. Also, higher recoveries resulted from painted concrete compared to wood, consistent with historical data on these materials. The highest decontamination efficacies were observed on the spores subjected to the longest exposures (48 hours) on both materials, with efficacies that gradually decreased with shorter exposures. Adsorption or absorption of the formaldehyde vapor may have been a factor, especially during the low temperature trials, resulting in less available formaldehyde in the air when measured. Conclusion: Environmental conditions affect formaldehyde concentrations in the air and thereby affect decontamination efficacy. Efficacy is also impacted by the material with which the contaminants are in contact.

The use of ozone gas for the inactivation of Bacillus anthracis and Bacillus subtilis spores on building materials.

A study was conducted to assess the efficacy of ozone gas in inactivating spores of both Bacillus anthracis and Bacillus subtilis inoculated onto six building materials (glass, wood, carpet, laminate, galvanized metal, and wallboard paper). Testing conditions consisted of ozone gas concentrations ranging from 7,000-12,000 parts per million (ppm), contact times from 4 to 12 h, and two relative humidity (RH) levels of 75 and 85%. Results showed that increasing the ozone concentration, contact time, and RH generally increased decontamination efficacy. The materials in which the highest decontamination efficacy was achieved for B. anthracis spores were wallboard paper, carpet, and wood with ≥ 6 log10 reduction (LR) occurring with 9,800 ppm ozone, 85% RH, for 6 h. The laminate and galvanized metal materials were generally more difficult to decontaminate, requiring 12,000 ppm ozone, 85% RH, and 9-12 h contact time to achieve ≥6 LR of B. anthracis. Lastly, overall, there were no significant differences in decontamination efficacy between the two species.

Evaluating the Environmental Persistence and Inactivation of MS2 Bacteriophage and the Presumed Ebola Virus Surrogate Phi6 Using Low Concentration Hydrogen Peroxide Vapor.

Ebola virus (EBOV) disease outbreaks, as well as the ability of EBOV to persist in the environment under certain conditions, highlight the need to develop effective decontamination techniques against the virus. We evaluated the efficacy of hydrogen peroxide vapor (HPV) to inactivate MS2 and Phi6 bacteriophages, the latter a recommended surrogate for EBOV. The phages were inoculated onto six material types with and without the presence of whole human blood. The inoculated materials were then exposed to either a high or low concentration of HPV for various elapsed times. The phages were also recovered from positive controls at these same elapsed times, to assess environmental persistence and decontamination efficacy. Low concentration hydrogen peroxide vapor (LCHP; 25 ppm) was effective against both phages on all materials without the presence of blood at 2 h. LCHP was ineffective against the phages in the presence of blood, on all materials, even with a 3-day contact time. Higher concentrations of HPV (>400 ppm) with contact times of 24-32 h achieved approximately 2-6 log reduction of the phages in the presence of blood.

No Amikacin, No Problem: a Successful Treatment Approach for Pediatric Otomastoiditis Due to Amikacin-Resistant Mycobacterium abscessus.

Pediatric otomastoiditis due to Mycobacterium abscessus can be a devastating infection that presents multiple treatment challenges. Van Wijk et al. report the use of a structured approach with a standardized regimen of intravenous (i.v.), topical, and oral antibiotics and surgical intervention for four pediatric patients with multidrug-resistant Mycobacterium abscessus otomastoiditis. Despite treatment-related adverse events, all four patients achieved sustained cure with this approach. This case series provides a framework for managing drug-resistant Mycobacterium abscessus otomastoiditis.

Native internally calibrated chromatin immunoprecipitation for quantitative studies of histone post-translational modifications.

Chromatin immunoprecipitation coupled to next-generation sequencing (ChIP-seq) has served as the central method for the study of histone modifications for the past decade. In ChIP-seq analyses, antibodies selectively capture nucleosomes bearing a modification of interest and the associated DNA is then mapped to the genome to determine the distribution of the mark. This approach has several important drawbacks: (i) ChIP interpretation necessitates the assumption of perfect antibody specificity, despite growing evidence that this is often not the case. (ii) Common methods for evaluating antibody specificity in other formats have little or no bearing on specificity within a ChIP experiment. (iii) Uncalibrated ChIP is reported as relative enrichment, which is biologically meaningless outside the experimental reference frame defined by a discrete immunoprecipitation (IP), thus preventing facile comparison across experimental conditions or modifications. (iv) Differential library amplification and loading onto next-generation sequencers, as well as computational normalization, can further compromise quantitative relationships that may exist between samples. Consequently, the researcher is presented with a series of potential pitfalls and is blind to nearly all of them. Here we provide a detailed protocol for internally calibrated ChIP (ICeChIP), a method we recently developed to resolve these problems by spike-in of defined nucleosomal standards within a ChIP procedure. This protocol is optimized for specificity and quantitative power, allowing for measurement of antibody specificity and absolute measurement of histone modification density (HMD) at genomic loci on a biologically meaningful scale enabling unambiguous comparisons. We provide guidance on optimal conditions for next-generation sequencing (NGS) and instructions for data analysis. This protocol takes between 17 and 18 h, excluding time for sequencing or bioinformatic analysis. The ICeChIP procedure enables accurate measurement of histone post-translational modifications (PTMs) genome-wide in mammalian cells as well as Drosophila melanogaster and Caenorhabditis elegans, indicating suitability for use in eukaryotic cells more broadly.

Influence of environmental conditions on the attenuation of ricin toxin on surfaces.

Ricin is a highly-toxic compound derived from castor plant beans. Several incidents involving contamination of residences and buildings due to ricin production or dissemination have occurred in recent years. The goal of this study was to determine whether ricin bioactivity could be attenuated in reasonable time via simple modifications of the indoor environment. Attenuation was assessed on six different materials as a function of temperature, relative humidity (RH), and contact time, using both a pure and crude preparation of the toxin. Ricin bioactivity was quantified via a cytotoxicity assay, and attenuation determined as the difference in ricin recovered from test and positive controls. The results showed that pure ricin could be attenuated successfully, while the crude ricin was generally more persistent and results more variable. We found no significant attenuation in crude ricin after two weeks at typical indoor environmental conditions, except on steel. Attenuation mostly improved with increasing temperature, but the effect of RH varied. For pure ricin, heat treatments at 40°C for 5 days or 50°C for 2-3 days achieved greater than 96% attenuation on steel. In contrast, appreciable recovery of the crude ricin preparation still occurred at 40°C after two weeks.