Application of the Draft NIOSH Occupational Exposure Banding Process to Bisphenol A: A case study.

Bisphenol A is a commercially important chemical used to make polycarbonate plastic, epoxy resins, and other specialty products. Despite an extensive body of in vitro, animal and human observational studies on the effects of exposure to bisphenol A, no authoritative bodies in the U.S. have adopted or recommended occupational exposure limits for bisphenol A. In 2017, the National Institute for Occupational Safety and Health published a Draft process for assigning health-protective occupational exposure bands, i.e., an airborne concentration range, to chemicals lacking an occupational exposure limit. Occupational exposure banding is a systematic process that uses both quantitative and qualitative toxicity information on selected health effect endpoints to assign an occupational exposure band for a chemical. The Draft process proposes three methodological tiers of increasing complexity for assigning an occupational exposure band. We applied Tier 1 (based on the Globally Harmonized System of Classification and Labelling) and Tier 2 (based on authoritative sources/reviews) to assign an occupational exposure band to bisphenol A. Under both Tier 1 and 2, the occupational exposure band for bisphenol A was "E" (<0.01 mg/m3), an assignment based on eye damage. "E" is the lowest exposure concentration range, reserved for chemicals with high potential toxicity. If eye damage was excluded in assigning an air concentration exposure range, then bisphenol A would band as "D" (>0.01 to 0.1 mg/m3) under Tier 1 (based on reproductive toxicity and respiratory/skin sensitization) and under Tier 2 (based on specific target organ toxicity-repeated exposure). In summary, Tiers 1 and 2 gave the same occupational exposure band for bisphenol A when eye damage was included ("E") or excluded ("D") as an endpoint.

Encapsidated hepatitis B virus reverse transcriptase is poised on an ordered RNA lattice.

Assembly of a hepatitis B virus (HBV) virion begins with the formation of an RNA-filled core composed of a symmetrical capsid (built of core protein), viral pregenomic RNA, and viral reverse transcriptase. To generate the circular dsDNA genome of HBV, reverse transcription requires multiple template switches within the confines of the capsid. To date, most anti-HBV therapeutics target this reverse transcription process. The detailed molecular mechanisms of this crucial process are poorly understood because of the lack of structural information. We hypothesized that capsid, RNA, and viral reverse transcriptase would need a precise geometric organization to accomplish reverse transcription. Here we present the asymmetric structure of authentic RNA-filled cores, determined to 14.5-Å resolution from cryo-EM data. Capsid and RNA are concentric. On the interior of the RNA, we see a distinct donut-like density, assigned to viral reverse transcriptase, which pins the viral pregenomic RNA to the capsid inner surface. The observation of a unique ordered structure inside the core suggests that assembly and the first steps of reverse transcription follow a single, determinate pathway and strongly suggests that all subsequent steps in DNA synthesis do as well.

Use of the "Exposome" in the Practice of Epidemiology: A Primer on -Omic Technologies.

The exposome has been defined as the totality of exposures individuals experience over the course of their lives and how those exposures affect health. Three domains of the exposome have been identified: internal, specific external, and general external. Internal factors are those that are unique to the individual, and specific external factors include occupational exposures and lifestyle factors. The general external domain includes sociodemographic factors such as educational level and financial status. Eliciting information on the exposome is daunting and not feasible at present; the undertaking may never be fully realized. A variety of tools have been identified to measure the exposome. Biomarker measurements will be one of the major tools in exposomic studies. However, exposure data can also be obtained from other sources such as sensors, geographic information systems, and conventional tools such as survey instruments. Proof-of-concept studies are being conducted that show the promise of exposomic investigation and the integration of different kinds of data. The inherent value of exposomic data in epidemiologic studies is that they can provide greater understanding of the relationships among a broad range of chemical and other risk factors and health conditions and ultimately lead to more effective and efficient disease prevention and control.

Insight into the mechanism of inhibition of adeno-associated virus by the Mre11/Rad50/Nbs1 complex.

UNLABELLED: Adeno-associated virus (AAV) is a dependent virus of the family Parvoviridae. The gene expression and replication of AAV and derived recombinant AAV (rAAV) vectors are severely limited (>10-fold) by the cellular DNA damage-sensing complex made up of Mre11, Rad50, and Nbs1 (MRN). The AAV genome does not encode the means to circumvent this block to productive infection but relies on coinfecting helper virus to do so. Using adenovirus helper proteins E1B55k and E4orf6, which enhance the transduction of AAV via degradation of MRN, we investigated the mechanism through which this DNA damage complex inhibits gene expression from rAAV. We tested the substrate specificity of inhibition and the contribution of different functions of the MRN complex. Our results demonstrate that both single- and double-stranded rAAV vectors are inhibited by MRN, which is in contrast to the predominant model that inhibition is the result of a block to second-strand synthesis. Exploring the contribution of known functions of MRN, we found that inhibition of rAAV does not require downstream DNA damage response factors, including signaling kinases ATM and ATR. The nuclease domain of Mre11 appears to play only a minor role in inhibition, while the DNA binding domain makes a greater contribution. Additionally, mutation of the inverted terminal repeat of the rAAV genome, which has been proposed to be the signal for interaction with MRN, is tolerated by the mechanism of inhibition. These results articulate a model of inhibition of gene expression in which physical interaction is more important than enzymatic activity and several key downstream damage repair factors are dispensable. IMPORTANCE: Many viruses modulate the host DNA damage response (DDR) in order to create a cellular environment permissive for infection. The MRN complex is a primary sensor of damage in the cell but also responds to invading viral genomes, often posing a block to infection. AAV is greatly inhibited by MRN and dependent on coinfecting helper virus, such as adenovirus, to remove this factor. Currently, the mechanism through which MRN inhibits AAV and other viruses is poorly understood. Our results reform the predominant model that inhibition of rAAV by MRN is due to limiting second-strand DNA synthesis. Instead, a novel mechanism of inhibition of gene expression independent of a block in rAAV DNA synthesis or downstream damage factors is indicated. These findings have clear implications for understanding this restriction to transduction of AAV and rAAV vectors, which have high therapeutic relevance and likely translate to other viruses that must navigate the DDR.

Enhanced transgene expression from recombinant single-stranded D-sequence-substituted adeno-associated virus vectors in human cell lines in vitro and in murine hepatocytes in vivo.

UNLABELLED: We have previously reported that the removal of a 20-nucleotide sequence, termed the D sequence, from both ends of the inverted terminal repeats (ITRs) in the adeno-associated virus serotype 2 (AAV2) genome significantly impairs rescue, replication, and encapsidation of the viral genomes (X. S. Wang, S. Ponnazhagan, and A. Srivastava, J Mol Biol 250:573-580, 1995; X. S. Wang, S. Ponnazhagan, and A. Srivastava, J Virol 70:1668-1677, 1996). Here we describe that replacement of only one D sequence in either ITR restores each of these functions, but DNA strands of only single polarity are encapsidated in mature progeny virions. Since most commonly used recombinant AAV vectors contain a single-stranded DNA (ssDNA), which is transcriptionally inactive, efficient transgene expression from AAV vectors is dependent upon viral second-strand DNA synthesis. We have also identified a transcription suppressor sequence in one of the D sequences, which shares homology with the binding site for the cellular NF-κB-repressing factor (NRF). The removal of this D sequence from, and replacement with a sequence containing putative binding sites for transcription factors in, single-stranded AAV (ssAAV) vectors significantly augments transgene expression both in human cell lines in vitro and in murine hepatocytes in vivo. The development of these genome-modified ssAAV vectors has implications not only for the basic biology of AAV but also for the optimal use of these vectors in human gene therapy. IMPORTANCE: The results of the studies described here not only have provided novel insights into some of the critical steps in the life cycle of a human virus, the adeno-associated virus (AAV), that causes no known disease but have also led to the development of novel recombinant AAV vectors which are more efficient in allowing increased levels of gene expression. Thus, these studies have significant implications for the potential use of these novel AAV vectors in human gene therapy.

Occupational safety and health, green chemistry, and sustainability: a review of areas of convergence.

With increasing numbers and quantities of chemicals in commerce and use, scientific attention continues to focus on the environmental and public health consequences of chemical production processes and exposures. Concerns about environmental stewardship have been gaining broader traction through emphases on sustainability and "green chemistry" principles. Occupational safety and health has not been fully promoted as a component of environmental sustainability. However, there is a natural convergence of green chemistry/sustainability and occupational safety and health efforts. Addressing both together can have a synergistic effect. Failure to promote this convergence could lead to increasing worker hazards and lack of support for sustainability efforts. The National Institute for Occupational Safety and Health has made a concerted effort involving multiple stakeholders to anticipate and identify potential hazards associated with sustainable practices and green jobs for workers. Examples of potential hazards are presented in case studies with suggested solutions such as implementing the hierarchy of controls and prevention through design principles in green chemistry and green building practices. Practical considerations and strategies for green chemistry, and environmental stewardship could benefit from the incorporation of occupational safety and health concepts which in turn protect affected workers.

Viral vectors for gene delivery to the central nervous system.

The potential benefits of gene therapy for neurological diseases such as Parkinson's, Amyotrophic Lateral Sclerosis (ALS), Epilepsy, and Alzheimer's are enormous. Even a delay in the onset of severe symptoms would be invaluable to patients suffering from these and other diseases. Significant effort has been placed in developing vectors capable of delivering therapeutic genes to the CNS in order to treat neurological disorders. At the forefront of potential vectors, viral systems have evolved to efficiently deliver their genetic material to a cell. The biology of different viruses offers unique solutions to the challenges of gene therapy, such as cell targeting, transgene expression and vector production. It is important to consider the natural biology of a vector when deciding whether it will be the most effective for a specific therapeutic function. In this review, we outline desired features of the ideal vector for gene delivery to the CNS and discuss how well available viral vectors compare to this model. Adeno-associated virus, retrovirus, adenovirus and herpesvirus vectors are covered. Focus is placed on features of the natural biology that have made these viruses effective tools for gene delivery with emphasis on their application in the CNS. Our goal is to provide insight into features of the optimal vector and which viral vectors can provide these features.

Roles of the envelope proteins in the amplification of covalently closed circular DNA and completion of synthesis of the plus-strand DNA in hepatitis B virus.

Covalently closed circular DNA (cccDNA), the nuclear form of hepatitis B virus (HBV), is synthesized by repair of the relaxed circular (RC) DNA genome. Initially, cccDNA is derived from RC DNA from the infecting virion, but additional copies of cccDNA are derived from newly synthesized RC DNA molecules in a process termed intracellular amplification. It has been shown that the large viral envelope protein limits the intracellular amplification of cccDNA for duck hepatitis B virus. The role of the envelope proteins in regulating the amplification of cccDNA in HBV is not well characterized. The present report demonstrates regulation of synthesis of cccDNA by the envelope proteins of HBV. Ablation of expression of the envelope proteins led to an increase (>6-fold) in the level of cccDNA. Subsequent restoration of envelope protein expression led to a decrease (>50%) in the level of cccDNA, which inversely correlated with the level of the envelope proteins. We found that the expression of L protein alone or in combination with M and/or S proteins led to a decrease in cccDNA levels, indicating that L contributes to the regulation of cccDNA. Coexpression of L and M led to greater regulation than either L alone or L and S. Coexpression of all three envelope proteins was also found to limit completion of plus-strand DNA synthesis, and the degree of this effect correlated with the level of the proteins and virion secretion.

Expanding control banding for workplace silica exposures throughout the Americas.

BACKGROUND: Silicosis, a lung disease caused by inhaling respirable crystalline silica dust, is an occupational illness affecting millions of workers worldwide. The National Institute for Occupational Safety and Health (NIOSH) has partnered with the World Health Organization, the International Labour Organization, and multiple agencies in the Americas to implement the program "The Elimination of Silicosis in the Americas". OBJECTIVES: One component of this program is control banding, a qualitative risk assessment and management strategy that allows non-experts to use task-based hazard data and potential exposure information to determine appropriate controls. RESULTS: From 2005 to the present, NIOSH occupational health researchers have worked with experts in Chile, Peru, Colombia, and Brazil to assess, implement, and provide tools to evaluate the use of control banding methodology.

A conformational switch in the DiGIR1 ribozyme involved in release and folding of the downstream I-DirI mRNA.

DiGIR1 is a group I-like cleavage ribozyme found as a structural domain within a nuclear twin-ribozyme group I intron. DiGIR1 catalyzes cleavage by branching at an Internal Processing Site (IPS) leading to formation of a lariat cap at the 5'-end of the 3'-cleavage product. The 3'-cleavage product is subsequently processed into an mRNA encoding a homing endonuclease. By analysis of combinations of 5'- and 3'-deletions, we identify a hairpin in the 5'-UTR of the mRNA (HEG P1) that is formed by conformational switching following cleavage. The formation of HEG P1 inhibits the reversal of the branching reaction, thus giving it directionality. Furthermore, the release of the mRNA is a consequence of branching rather than hydrolytic cleavage. A model is put forward that explains the release of the I-DirI mRNA with a lariat cap and a structured 5'-UTR as a direct consequence of the DiGIR1 branching reaction. The role of HEG P1 in GIR1 branching is reminiscent of that of hairpin P-1 in splicing of the Tetrahymena rRNA group I intron and illustrates a general principle in RNA-directed RNA processing.

The evolution of skin notations for occupational risk assessment: a new NIOSH strategy.

This article presents an overview of a strategy for assignment of hazard-specific skin notations (SK), developed by the National Institute for Occupational Safety and Health (NIOSH). This health hazard characterization strategy relies on multiple SKs capable of delineating systemic (SYS), direct (DIR), and immune-mediated (SEN) adverse effects caused by dermal exposures to chemicals. One advantage of the NIOSH strategy is the ability to combine SKs when it is determined that a chemical may cause multiple adverse effects following dermal contact (e.g., SK: SYS-DIR-SEN). Assignment of the SKs is based on a weight-of-evidence (WOE) approach, which refers to the critical examination of all available data from diverse lines of evidence and the derivation of a scientific interpretation based on the collective body of data including its relevance, quality, and reported results. Numeric cutoff values, based on indices of toxic potency, serve as guidelines to aid in consistently determining a chemical's relative toxicity and hazard potential. The NIOSH strategy documents the scientific rationale for determination of the hazard potential of a chemical and the subsequent assignment of SKs. A case study of acrylamide is presented as an application of the NIOSH strategy.

History of the Department of Cell Biology at Yale School of Medicine, 1813-2010.

The Department of Cell Biology at the Yale University School of Medicine was established in 1983. It was preceded by the Section of Cell Biology, which was formed in 1973 when George E. Palade and collaborators came to Yale from the Rockefeller University. Cell Biology at Yale had its origins in the Department of Anatomy that existed from the beginning of classes at the Medical Institution of Yale College in 1813. This article reviews the history of the Department of Anatomy at Yale and its evolution into Cell Biology that began with the introduction of histology into the curriculum in the 1860s. The formation and development of the Section and Department of Cell Biology in the second half of the 20th century to the present time are described. Biographies and research activities of the chairs and key faculty in anatomy and cell biology are provided.

Integrating research into a molecular cloning course to address the evolving biotechnology landscape.

The rapid development of molecular biotechnology presents a curricular challenge for educators trying to provide students with relevant coursework. A comprehensive biology education should also include opportunities for students to develop intellectual and technical skills through authentic research experiences. Integrating relevant and interesting research projects into their classes, however, can be a challenging task for instructors. To address these varied demands, we redesigned our existing molecular cloning course to incorporate an independent research project assessing calcium signaling. In the revised course, students use traditional and recombination-based cloning strategies to generate bacterial and mammalian expression vectors encoding CaMPARI, a novel fluorescent calcium indicator. Bacterially-expressed CaMPARI is used in protein quantification and purification assays. Students must also design their own research project evaluating the effect of chemotherapeutic agents on calcium signaling in a mammalian system. Revised and novel labs were designed to be modular, facilitating their integration into the course over 2 years. End-of-semester student evaluations were compared between years revealing a significant difference in students' perception of the course's difficulty between years. This change in attitude highlights potential pedagogical considerations that must be examined when introducing new material and activities into existing courses. Since calcium signaling is important for cellular process across diverse species, instructors may be able to develop research projects within their respective areas of interest. Integration of authentic research experiences into the curriculum is challenging; however, the framework described here provides a versatile structure that can be adapted to merge diverse instructor interests with evolving educational needs.

Transforming a PICU Into an Adult ICU During the Coronavirus Disease 2019 Pandemic: Meeting Multiple Needs.

We describe the process converting half of our 40-bed PICU into a negative-pressure biocontainment ICU dedicated to adult coronavirus disease 2019 patients within a 1,003-bed academic quaternary hospital. We outline the construction, logistics, supplies, provider education, staffing, and operations. We share lessons learned of working with a predominantly pediatric staff blended with adult expertise staff while maintaining elements of family-centered care typical of pediatric critical care medicine. Critically ill coronavirus disease 2019 adult patients may be cared for in a PICU and care may be augmented by implementing elements of holistic, family-centered PICU practice.

A framework for integrating information resources for chemical emergency management and response.

Effective emergency management and response require appropriate utilization of various resources as an incident evolves. This manuscript describes the information resources used in chemical emergency management and operations and how their utility evolves from the initial response phase to recovery to event close out. The authors address chemical hazard guidance in the context of four different phases of emergency response: preparedness, emergency response (both initial and ongoing), recovery, and mitigation. Immediately following a chemical incident, during the initial response, responders often use readily available, broad-spectrum guidance to make rapid decisions in the face of uncertainties regarding potential exposure to physical and health hazards. Physical hazards are described as the hazards caused by chemicals that can cause harm with or without direct contact. Examples of physical hazards include explosives, flammables, and gases under pressure. This first line of resources may not be chemical-specific in nature, but it can provide guidance related to isolation distances, protective actions, and the most important physical and health threats. During the ongoing response phase, an array of resources can provide detailed information on physical and health hazards related to specific chemicals of concern. Consequently, risk management and mitigation actions evolve as well. When the incident stabilizes to a recovery phase, the types of information resources that facilitate safe and effective incident management evolve. Health and physical concerns transition from acute toxicity and immediate hazards to both immediate and latent health effects. Finally, the information inputs utilized during the preparedness phase include response evaluations of past events, emergency preparedness planning, and chemical-specific guidance about chemicals present. This manuscript details a framework for identifying the effective use of information resources at each phase and provides case study examples from chemical hazard emergencies.

A framework for integrating information resources for chemical emergency management and response.

Effective emergency management and response require appropriate utilization of various resources as an incident evolves. This manuscript describes the information resources used in chemical emergency management and operations and how their utility evolves from the initial response phase to recovery to event close out. The authors address chemical hazard guidance in the context of four different phases of emergency response: preparedness, emergency response (both initial and ongoing), recovery, and mitigation. Immediately following a chemical incident, during the initial response, responders often use readily available, broad-spectrum guidance to make rapid decisions in the face of uncertainties regarding potential exposure to physical and health hazards. Physical hazards are described as the hazards caused by chemicals that can cause harm with or without direct contact. Examples of physical hazards include explosives, flammables, and gases under pressure. This first line of resources may not be chemical-specific in nature, but it can provide guidance related to isolation distances, protective actions, and the most important physical and health threats. During the ongoing response phase, an array of resources can provide detailed information on physical and health hazards related to specific chemicals of concern. Consequently, risk management and mitigation actions evolve as well. When the incident stabilizes to a recovery phase, the types of information resources that facilitate safe and effective incident management evolve. Health and physical concerns transition from acute toxicity and immediate hazards to both immediate and latent health effects. Finally, the information inputs utilized during the preparedness phase include response evaluations of past events, emergency preparedness planning, and chemical-specific guidance about chemicals present. This manuscript details a framework for identifying the effective use of information resources at each phase and provides case study examples from chemical hazard emergencies.

Characterization of a novel pathogenic variant in the FECH gene associated with erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) is an autosomal recessive deficiency in heme biosynthesis due to pathogenic variants in the ferrochelatase gene (FECH). Patients present with lifelong photosensitivity and potential liver disease. Here we report a novel FECH variant designated c.904_912+1del found in trans with the c.315-48T>C hypomorphic variant, in one family with three affected individuals. These patients presented with immediate painful cutaneous photosensitivity but no hepatic manifestations. All have elevated protoporphyrin levels consistent with a diagnosis of EPP. Genetic, biochemical, and functional assay results obtained for this family suggest that the unique variant c.904_912+1del is likely pathogenic and thus causative of EPP.

Unique Down to Our Microbes-Assessment of an Inquiry-Based Metagenomics Activity.

Metagenomics is an important method for studying microbial life. However, undergraduate exposure to metagenomics is hindered by associated software, computing demands, and dataset access. In this inquiry-based activity designed for introductory life science majors and nonmajors, students perform an investigation of the bacterial communities inhabiting the human belly button and associated metagenomics data collected through a citizen science project and visualized using an open-access bioinformatics tool. The activity is designed for attainment of the following student learning outcomes: defining terms associated with metagenomics analyses, describing the biological impact of the microbiota on human health, formulating a hypothesis, analyzing and interpreting metagenomics data to compare microbiota, evaluating a specific hypothesis, and synthesizing a conceptual model as to why bacterial populations vary. This activity was implemented in six introductory biology and biotechnology courses across five institutions. Attainment of student learning outcomes was assessed through completion of a quiz and students' presentations of their findings. In presentations, students demonstrated their ability to develop novel hypotheses and analyze and interpret metagenomic data to evaluate their hypothesis. In quizzes, students demonstrated their ability to define key terms and describe the biological impact of the microbiota on human health. Student learning gains assessment also revealed that students perceived gains for all student learning outcomes. Collectively, our assessment demonstrates achievement of the learning outcomes and supports the utility of this inquiry-based activity to engage undergraduates in the scientific process via analyses of metagenomics datasets and associated exploration of a microbial community that lives on the human body.

The characterization of airborne occupational safety and health hazards in selected small businesses; manufacturing wood pallets.

Researchers from the National Institute for Occupational Safety and Health (NIOSH) investigated occupational safety and health concerns in the small business wood pallet manufacturing industry because of an injury rate (2000) 226% greater than that for general industry. NIOSH investigators conducted walk-through evaluations at seven wood pallet manufacturing companies, and returned to four of them to take environmental measurements. Carbon monoxide (CO) levels, noise levels, and total particulate were measured, ergonomic observations made, and occupational safety practices analyzed at each of the four facilities where measurements were taken. The focus of this study is the evaluation of airborne particulate and carbon monoxide exposures for the purpose of determining areas of potentially high exposures. This knowledge can guide the plant owner or health professional to determine whether further measurements are necessary and where they might be needed. Safety factors and physical stressors (noise and ergonomic stressors) were described in a previously published companion paper. Although we did not take 8 h samples, we did find certain exposures that were potentially of concern to the small business owner. The main findings of this investigation were as follows: 1) CO levels in three plants, for the time periods measured, were less than the OSHA permissible exposure limit (PEL) of 50 parts per million (ppm) for an 8-h TWA. Three measurements, all from one plant, were due to a older and defective forklift and were above 50 ppm. 2) Total dust measures ranged from 0.86 to 1.67 mg/m3, taken adjacent to an operating machine cutting hardwood and measured up to 6 min. The American Conference of Governmental Industrial Hygienists (ACGIH) guideline for hardwood dust is 1.0 mg/m3, again for an 8-h TWA.

Recombinant adeno-associated virus: clinical application and development as a gene-therapy vector.

Gene therapy is gaining momentum as a method of treating human disease. Initially conceived as a strategy to complement defective genes in monogenic disorders, the scope of gene therapy has expanded to encompass a variety of applications. Likewise, the molecular tools for gene delivery have evolved and diversified to meet these various therapeutic needs. Recombinant adeno-associated virus (rAAV) has made significant strides toward clinical application with an excellent safety profile and successes in several clinical trials. This review covers the basic biology of rAAV as a gene therapy vector as well as its advantages compared with other methods of gene delivery. The status of clinical trials utilizing rAAV is also discussed in detail. In conclusion, methods of engineering the vector to overcome challenges identified from these trials are covered, with emphasis on modification of the viral capsid to increase the tissue/cell-specific targeting and transduction efficiency.