CDC Deployments to State, Tribal, Local, and Territorial Health Departments for COVID-19 Emergency Public Health Response - United States, January 21-July 25, 2020.

Coronavirus disease 2019 (COVID-19) is a viral respiratory illness caused by SARS-CoV-2. During January 21-July 25, 2020, in response to official requests for assistance with COVID-19 emergency public health response activities, CDC deployed 208 teams to assist 55 state, tribal, local, and territorial health departments. CDC deployment data were analyzed to summarize activities by deployed CDC teams in assisting state, tribal, local, and territorial health departments to identify and implement measures to contain SARS-CoV-2 transmission (1). Deployed teams assisted with the investigation of transmission in high-risk congregate settings, such as long-term care facilities (53 deployments; 26% of total), food processing facilities (24; 12%), correctional facilities (12; 6%), and settings that provide services to persons experiencing homelessness (10; 5%). Among the 208 deployed teams, 178 (85%) provided assistance to state health departments, 12 (6%) to tribal health departments, 10 (5%) to local health departments, and eight (4%) to territorial health departments. CDC collaborations with health departments have strengthened local capacity and provided outbreak response support. Collaborations focused attention on health equity issues among disproportionately affected populations (e.g., racial and ethnic minority populations, essential frontline workers, and persons experiencing homelessness) and through a place-based focus (e.g., persons living in rural or frontier areas). These collaborations also facilitated enhanced characterization of COVID-19 epidemiology, directly contributing to CDC data-informed guidance, including guidance for serial testing as a containment strategy in high-risk congregate settings, targeted interventions and prevention efforts among workers at food processing facilities, and social distancing.

Career orientation and perceived professional competence among clinical research coordinators.

INTRODUCTION: This study identified underlying career orientation types of clinical research coordinators (CRCs) using cluster analysis. Select career (satisfaction, engagement, and planning) and competency-related (perceived competence) information was used to identify four distinct career orientation types. METHOD: A web-based survey was administered to CRCs employed in one of four research institutions affiliated with a National Institutes of Health-funded Clinical and Translational Research Award (CTSA) in the southeastern USA. Each respondent completed a survey containing questions about personal background, individual attributes, perceived professional competence, and career orientation. RESULTS: The first CRC type (35.2%) possessed a positive, knowledge-seeking orientation, characterized by high career-related scores but a conservative assessment of perceived competence. The second CRC type (18.6%) represented an optimistic and confident career orientation reflected in moderate to high scores on each of the four identifying factors. The third CRC type (27.6%) reflected an inconsistent career orientation highlighted by lowered perceived competence. The final CRC type (18.6%) reflected a disengaged orientation characterized by negative responses to all career and competence factors. CONCLUSION: Understanding the career orientation of CRCs can be helpful to institutional administrators and clinical investigators as they seek to support the professional development of CRCs through tailored training efforts or work-related supports. Knowledge of career orientation may also inform individual CRCs as they manage their personal career paths by assessing current levels of functioning, career-related strengths or weaknesses, and training needs.

Progeny Varicella-Zoster Virus Capsids Exit the Nucleus but Never Undergo Secondary Envelopment during Autophagic Flux Inhibition by Bafilomycin A1.

Varicella-zoster virus (VZV) is an alphaherpesvirus that lacks the herpesviral neurovirulence protein ICP34.5. The underlying hypothesis of this project was that inhibitors of autophagy reduce VZV infectivity. We selected the vacuolar proton ATPase inhibitor bafilomycin A1 for analysis because of its well-known antiautophagy property of impeding acidification during the late stage of autophagic flux. We documented that bafilomycin treatment from 48 to 72 h postinfection lowered VZV titers substantially (P ≤ 0.008). Because we were unable to define the site of the block in the infectious cycle by confocal microscopy, we turned to electron microscopy. Capsids were observed in the nucleus, in the perinuclear space, and in the cytoplasm adjacent to Golgi apparatus vesicles. Many of the capsids had an aberrant appearance, as has been observed previously in infections not treated with bafilomycin. In contrast to prior untreated infections, however, secondary envelopment of capsids was not seen in the trans-Golgi network, nor were prototypical enveloped particles with capsids (virions) seen in cytoplasmic vesicles after bafilomycin treatment. Instead, multiple particles with varying diameters without capsids (light particles) were seen in large virus assembly compartments near the disorganized Golgi apparatus. Bafilomycin treatment also led to increased numbers of multivesicular bodies in the cytoplasm, some of which contained remnants of the Golgi apparatus. In summary, we have defined a previously unrecognized property of bafilomycin whereby it disrupted the site of secondary envelopment of VZV capsids by altering the pH of the trans-Golgi network and thereby preventing the correct formation of virus assembly compartments.IMPORTANCE This study of VZV assembly in the presence of bafilomycin A1 emphasizes the importance of the Golgi apparatus/trans-Golgi network as a platform in the alphaherpesvirus life cycle. We have previously shown that VZV induces levels of autophagy far above the basal levels of autophagy in human skin, a major site of VZV assembly. The current study documented that bafilomycin treatment led to impaired assembly of VZV capsids after primary envelopment/de-envelopment but before secondary reenvelopment. This VZV study also complemented prior herpes simplex virus 1 and pseudorabies virus studies investigating two other inhibitors of endoplasmic reticulum (ER)/Golgi apparatus function: brefeldin A and monensin. Studies with porcine herpesvirus demonstrated that primary enveloped particles accumulated in the perinuclear space in the presence of brefeldin A, while studies with herpes simplex virus 1 documented an impaired secondary assembly of enveloped viral particles in the presence of monensin.

Neuroprotective levels of IGF-1 exacerbate epileptogenesis after brain injury.

Exogenous Insulin-Like Growth Factor-1 (IGF-1) is neuroprotective in animal models of brain injury, and has been considered as a potential therapeutic. Akt-mTOR and MAPK are downstream targets of IGF-1 signaling that are activated after brain injury. However, both brain injury and mTOR are linked to epilepsy, raising the possibility that IGF-1 may be epileptogenic. Here, we considered the role of IGF-1 in development of epilepsy after brain injury, using the organotypic hippocampal culture model of post-traumatic epileptogenesis. We found that IGF-1 was neuroprotective within a few days of injury but that long-term IGF-1 treatment was pro-epileptic. Pro-epileptic effects of IGF-1 were mediated by Akt-mTOR signaling. We also found that IGF-1 - mediated increase in epileptic activity led to neurotoxicity. The dualistic nature of effects of IGF-1 treatment demonstrates that anabolic enhancement through IGF-1 activation of mTOR cascade can be beneficial or harmful depending on the stage of the disease. Our findings suggest that epilepsy risk may need to be considered in the design of neuroprotective treatments for brain injury.

Wnt Signaling Regulates Airway Epithelial Stem Cells in Adult Murine Submucosal Glands.

Wnt signaling is required for lineage commitment of glandular stem cells (SCs) during tracheal submucosal gland (SMG) morphogenesis from the surface airway epithelium (SAE). Whether similar Wnt-dependent processes coordinate SC expansion in adult SMGs following airway injury remains unknown. We found that two Wnt-reporters in mice (BAT-gal and TCF/Lef:H2B-GFP) are coexpressed in actively cycling SCs of primordial glandular placodes and in a small subset of adult SMG progenitor cells that enter the cell cycle 24 hours following airway injury. At homeostasis, these Wnt reporters showed nonoverlapping cellular patterns of expression in the SAE and SMGs. Following tracheal injury, proliferation was accompanied by dynamic changes in Wnt-reporter activity and the analysis of 56 Wnt-related signaling genes revealed unique temporal changes in expression within proximal (gland-containing) and distal (gland-free) portions of the trachea. Wnt stimulation in vivo and in vitro promoted epithelial proliferation in both SMGs and the SAE. Interestingly, slowly cycling nucleotide label-retaining cells (LRCs) of SMGs were spatially positioned near clusters of BAT-gal positive serous tubules. Isolation and culture of tet-inducible H2B-GFP LRCs demonstrated that SMG LRCs were more proliferative than SAE LRCs and culture expanded SMG-derived progenitor cells outcompeted SAE-derived progenitors in regeneration of tracheal xenograft epithelium using a clonal analysis competition assay. SMG-derived progenitors were also multipotent for cell types in the SAE and formed gland-like structures in xenografts. These studies demonstrate the importance of Wnt signals in modulating SC phenotypes within tracheal niches and provide new insight into phenotypic differences of SMG and SAE SCs. Stem Cells 2016;34:2758-2771.

Differential cytotoxicity of long-chain bases for human oral gingival epithelial keratinocytes, oral fibroblasts, and dendritic cells.

Long-chain bases, found in the oral cavity, have potent antimicrobial activity against oral pathogens. In an article associated with this dataset, Poulson and colleagues determined the cytotoxicities of long-chain bases (sphingosine, dihydrosphingosine, and phytosphingosine) for human oral gingival epithelial (GE) keratinocytes, oral gingival fibroblasts (GF), dendritic cells (DC), and squamous cell carcinoma (SCC) cell lines [1]. Poulson and colleagues found that GE keratinocytes were more resistant to long-chain bases as compared to GF, DC, and SCC cell lines [1]. In this study, we assess the susceptibility of DC to lower concentrations of long chain bases. 0.2-10.0 µM long-chain bases and GML were not cytotoxic to DC; 40.0-80.0 µM long-chain bases, but not GML, were cytotoxic for DC; and 80.0 µM long-chain bases were cytotoxic to DC and induced cellular damage and death in less than 20 mins. Overall, the LD50 of long-chain bases for GE keratinocytes, GF, and DC were considerably higher than their minimal inhibitory concentrations for oral pathogens, a finding important to pursuing their future potential in treating periodontal and oral infections.

CD151 promotes α3ß1 integrin-dependent organization of carcinoma cell junctions and restrains collective cell invasion.

Integrins function in collective migration both as major receptors for extracellular matrix and by crosstalk to adherens junctions. Despite extensive research, important questions remain about how integrin signaling mechanisms are integrated into collective migration programs. Tetraspanins form cell surface complexes with a subset of integrins and thus are good candidates for regulating the balance of integrin functional inputs into cell-matrix and cell-cell interactions. For example, tetraspanin CD151 directly associates with α3ß1 integrin in carcinoma cells and promotes rapid α3ß1-dependent single cell motility, but CD151 also promotes organized adherens junctions and restrains collective carcinoma cell migration on 2D substrates. However, the individual roles of CD151s integrin partners in CD151s pro-junction activity in carcinoma cells were not well understood. Here we find that CD151 promotes organized carcinoma cell junctions via α3ß1 integrin, by a mechanism that requires the a3b1 ligand, laminin-332. Loss of CD151 promotes collective 3D invasion and growth in vitro and in vivo, and the enhanced invasion of CD151-silenced cells is α3 integrin dependent, suggesting that CD151 can regulate the balance between α3ß1s pro-junction and pro-migratory activities in collective invasion. An analysis of human cancer cases revealed that changes in CD151 expression can be linked to either better or worse clinical outcomes depending on context, including potentially divergent roles for CD151 in different subsets of breast cancer cases. Thus, the role of the CD151-α3ß1 complex in carcinoma progression is context dependent, and may depend on the mode of tumor cell invasion.

Codon-optimized fluorescent mTFP and mCherry for microscopic visualization and genetic counterselection of streptococci and enterococci.

Despite the powerful potential of fluorescent proteins for labeling bacteria, their use has been limited in multi-species oral biofilm models. Fermentative metabolism by streptococcal species that initiate biofilm colonization results in an acidic, reduced microenvironment that may limit the activities of some fluorescent proteins which are influenced by pH and oxygen availability. The need to reliably distinguish morphologically similar strains within biofilms was the impetus for this work. Teal fluorescent protein (mTFP1) and red fluorescent protein (mCherry) were chosen because their fluorescent properties made them promising candidates. Since tRNA availability has been implicated in efficient translation of sufficient quantities of protein for maximum fluorescence, a streptococcal codon optimization approach was used. DNA was synthesized to encode either protein using codons most frequently used in streptococci; each coding region was preceded by an engineered ribosomal binding site and restriction sites for cloning a promoter. Plasmids carrying this synthesized DNA under control of the Streptococcus mutans lactate dehydrogenase promoter conferred fluorescence to nine representative streptococcal and two Enterococcus faecalis strains. Further characterization in Streptococcus gordonii showed that mTFP1 and mCherry expressions could be detected in cells grown planktonically, in biofilms, or in colonies on agar when expressed on an extrachromosomal plasmid or in single copy integrated into the chromosome. This latter property facilitated counterselection of chromosomal mutations demonstrating value for bacterial strain construction. Fluorescent and non-fluorescent bacteria were distinguishable at acidic pH. These codon-optimized versions of mTFP1 and mCherry have promising potential for use in multiple experimental applications.

Differential cytotoxicity of long-chain bases for human oral gingival epithelial keratinocytes, oral fibroblasts, and dendritic cells.

Long-chain bases are present in the oral cavity. Previously we determined that sphingosine, dihydrosphingosine, and phytosphingosine have potent antimicrobial activity against oral pathogens. Here, we determined the cytotoxicities of long-chain bases for oral cells, an important step in considering their potential as antimicrobial agents for oral infections. This information would clearly help in establishing prophylactic or therapeutic doses. To assess this, human oral gingival epithelial (GE) keratinocytes, oral gingival fibroblasts (GF), and dendritic cells (DC) were exposed to 10.0-640.0 µM long-chain bases and glycerol monolaurate (GML). The effects of long-chain bases on cell metabolism (conversion of resazurin to resorufin), membrane permeability (uptake of propidium iodide or SYTOX-Green), release of cellular contents (LDH), and cell morphology (confocal microscopy) were all determined. GE keratinocytes were more resistant to long-chain bases as compared to GF and DC, which were more susceptible. For DC, 0.2-10.0 µM long-chain bases and GML were not cytotoxic; 40.0-80.0 µM long-chain bases, but not GML, were cytotoxic; and 80.0 µM long-chain bases induced cellular damage and death in less than 20 min. The LD50 of long-chain bases for GE keratinocytes, GF, and DC were considerably higher than their minimal inhibitory concentrations for oral pathogens, a finding important to pursuing their future potential in treating periodontal and oral infections.

Charge, size, and cellular selectivity for multiwall carbon nanotubes by maize and soybean.

Maize (Zea mays) and soybean (Glycine max) were used as model food-chain plants to explore vegetative uptake of differently charged multiwall carbon nanotubes (MWCNTs). Three types of MWCNTs, including neutral pristine MWCNT (p-MWCNT), positively charged MWCNT-NH2, and negatively charged MWCNT-COOH, were directly taken-up and translocated from hydroponic solution to roots, stems, and leaves of maize and soybean plants at the MWCNT concentrations ranging from 10.0 to 50.0 mg/L during 18-day exposures. MWCNTs accumulated in the xylem and phloem cells and within specific intracellular sites like the cytoplasm, cell wall, cell membrane, chloroplast, and mitochondria, which was observed by transmission electron microscopy. MWCNTs stimulated the growth of maize and inhibited the growth of soybean at the exposed doses. The cumulative transpiration of water in maize exposed to 50 mg/L of MWCNT-COOHs was almost twice as much as that in the maize control. Dry biomass of maize exposed to MWCNTs was greater than that of maize control. In addition, the uptake and translocation of these MWCNTs clearly exhibited cellular, charge, and size selectivity in maize and soybean, which could be important properties for nanotransporters. This is the first report of cellular, charge, and size selectivity on the uptake by whole food plants for three differently charged MWCNTs.

Transport of gold nanoparticles through plasmodesmata and precipitation of gold ions in woody poplar.

Poplar plants (Populus deltoides × nigra, DN-34) were used as a model to explore vegetative uptake of commercially available gold nanoparticles (AuNPs) and their subsequent translocation and transport into plant cells. AuNPs were directly taken up and translocated from hydroponic solution to poplar roots, stems and leaves. Total gold concentrations in leaves of plants treated with 15, 25 and 50 nm AuNPs at exposure concentrations of 498±50.5, 247±94.5 and 263±157 ng/mL in solutions were: 0.023±0.006, 0.0218±0.004 and 0.005±0.0003 µg/g dry weight, respectively, which accounted for 0.05, 0.10 and 0.03%, respectively, of the total gold mass added. The presence of total gold in plant tissues was measured by inductively coupled plasma mass spectrometry, while AuNPs were observed by transmission electron microscopy in plant tissues. In solution, AuNPs were distinguished from Au(III) ions by membrane separation and centrifugation. AuNPs behaved conservatively inside the plants and were not dissolved into gold ions. On the other hand, Au(III) ions were taken up and reduced into AuNPs inside whole plants. AuNPs were observed in the cytoplasm and various organelles of root and leaf cells. A distinct change in color from yellow to pink was observed as Au(III) ions were reduced and precipitated in hydroponic solution. The accumulation of AuNPs in the plasmodesma of the phloem complex in root cells clearly suggests ease of transport between cells and translocation throughout the whole plant, inferring the potential for entry and transfer in food webs.

Oral mucosal lipids are antibacterial against Porphyromonas gingivalis, induce ultrastructural damage, and alter bacterial lipid and protein compositions.

Oral mucosal and salivary lipids exhibit potent antimicrobial activity for a variety of Gram-positive and Gram-negative bacteria; however, little is known about their spectrum of antimicrobial activity or mechanisms of action against oral bacteria. In this study, we examine the activity of two fatty acids and three sphingoid bases against Porphyromonas gingivalis, an important colonizer of the oral cavity implicated in periodontitis. Minimal inhibitory concentrations, minimal bactericidal concentrations, and kill kinetics revealed variable, but potent, activity of oral mucosal and salivary lipids against P. gingivalis, indicating that lipid structure may be an important determinant in lipid mechanisms of activity against bacteria, although specific components of bacterial membranes are also likely important. Electron micrographs showed ultrastructural damage induced by sapienic acid and phytosphingosine and confirmed disruption of the bacterial plasma membrane. This information, coupled with the association of treatment lipids with P. gingivalis lipids revealed via thin layer chromatography, suggests that the plasma membrane is a likely target of lipid antibacterial activity. Utilizing a combination of two-dimensional in-gel electrophoresis and Western blot followed by mass spectroscopy and N-terminus degradation sequencing we also show that treatment with sapienic acid induces upregulation of a set of proteins comprising a unique P. gingivalis stress response, including proteins important in fatty acid biosynthesis, metabolism and energy production, protein processing, cell adhesion and virulence. Prophylactic or therapeutic lipid treatments may be beneficial for intervention of infection by supplementing the natural immune function of endogenous lipids on mucosal surfaces.

Effects of Eyjafjallajökull volcanic ash on innate immune system responses and bacterial growth in vitro.

BACKGROUND: On 20 March 2010, the Icelandic volcano Eyjafjallajökull erupted for the first time in 190 years. Despite many epidemiological reports showing effects of volcanic ash on the respiratory system, there are limited data evaluating cellular mechanisms involved in the response to ash. Epidemiological studies have observed an increase in respiratory infections in subjects and populations exposed to volcanic eruptions. METHODS: We physicochemically characterized volcanic ash, finding various sizes of particles, as well as the presence of several transition metals, including iron. We examined the effect of Eyjafjallajökull ash on primary rat alveolar epithelial cells and human airway epithelial cells (20-100 µg/cm(2)), primary rat and human alveolar macrophages (5-20 µg/cm(2)), and Pseudomonas aeruginosa (PAO1) growth (3 µg/104 bacteria). RESULTS: Volcanic ash had minimal effect on alveolar and airway epithelial cell integrity. In alveolar macrophages, volcanic ash disrupted pathogen-killing and inflammatory responses. In in vitro bacterial growth models, volcanic ash increased bacterial replication and decreased bacterial killing by antimicrobial peptides. CONCLUSIONS: These results provide potential biological plausibility for epidemiological data that show an association between air pollution exposure and the development of respiratory infections. These data suggest that volcanic ash exposure, while not seriously compromising lung cell function, may be able to impair innate immunity responses in exposed individuals.

Human ß-defensin-3 alters, but does not inhibit, the binding of Porphyromonas gingivalis haemagglutinin B to the surface of human dendritic cells.

Human ß-defensin-3 (HBD3) is a small, cationic, host defence peptide with broad antimicrobial activities and diverse innate immune functions. HBD3 binds to many microbial antigens and, in this study, we hypothesised that the known binding of HBD3 to Porphyromonas gingivalis recombinant haemagglutinin B (rHagB) alters, but does not inhibit, the binding of rHagB to human dendritic cells. To test this, human myeloid dendritic cells were incubated for 5 min with rHagB, HBD3 + rHagB (10:1 molar ratio), HBD3 or 0.1 M phosphate-buffered saline (PBS) (pH 7.2) and were then rapidly fixed and processed for confocal microscopy and ultramicrotomy. rHagB and HBD3 could be detected with primary monoclonal mouse antibody to rHagB (MoAb 1858) or polyclonal rabbit antibody to HBD3 (P241) and secondary fluorescent-labelled anti-mouse or anti-rabbit antibodies (confocal microscopy) or protein A-colloidal gold (immunoelectron microscopy). In cells incubated with rHagB only, fluorescence and protein A-colloidal gold were seen at the cell surface and throughout the cytoplasm. In cells incubated with HBD3+rHagB, fluorescence was observed only at the cell surface in a 'string of pearls' configuration. Overall, these results suggest that HBD3 binding to rHagB alters, but does not inhibit, the binding of rHagB to human myeloid dendritic cells.

Identification of an autophagy defect in smokers' alveolar macrophages.

Alveolar macrophages are essential for clearing bacteria from the alveolar surface and preventing microbe-induced infections. It is well documented that smokers have an increased incidence of infections, in particular lung infections. Alveolar macrophages accumulate in smokers' lungs, but they have a functional immune deficit. In this study, we identify an autophagy defect in smokers' alveolar macrophages. Smokers' alveolar macrophages accumulate both autophagosomes and p62, a marker of autophagic flux. The decrease in the process of autophagy leads to impaired protein aggregate clearance, dysfunctional mitochondria, and defective delivery of bacteria to lysosomes. This study identifies the autophagy pathway as a potential target for interventions designed to decrease infection rates in smokers and possibly in individuals with high environmental particulate exposure.

Gene expression and microscopic analysis of Arabidopsis exposed to chloroacetanilide herbicides and explosive compounds. A phytoremediation approach.

Understanding the function of detoxifying enzymes in plants toward xenobiotics is of major importance for phytoremediation applications. In this work, Arabidopsis (Arabidopsis thaliana; ecotype Columbia) seedlings were exposed to 0.6 mm acetochlor (AOC), 2 mm metolachlor (MOC), 0.6 mm 2,4,6-trinitrotoluene (TNT), and 0.3 mm hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). In vivo glutathione (GSH) conjugation reactions of AOC, MOC, RDX, and TNT were studied in root cells using a multiphoton microscope. In situ labeling with monochlorobimane, used as a competitive compound for conjugation reactions with GSH, confirmed that AOC and MOC are conjugated in Arabidopsis cells. Reverse transcription-PCR established the expression profile of glutathione S-transferases (GSTs) and nitroreductases enzymes. Genes selected for this study were AtGSTF2, AtGSTU1, AtGSTU24, and two isoforms of 12-oxophytodienoate reductase (OPR1 and OPR2). The five transcripts tested were induced by all treatments, but RDX resulted in low induction. The mRNA level of AtGSTU24 showed substantial increase for all chemicals (23-fold induction for AOC, 18-fold for MOC, 5-fold for RDX, and 40-fold for TNT). It appears that GSTs are also involved in the conjugation reactions with metabolites of TNT, and to a lesser extent with RDX. Results indicate that OPR2 is involved in plant metabolism of TNT (11-fold induction), and in oxidative stress when exposed to AOC (7-fold), MOC (9-fold), and RDX (2-fold). This study comprises gene expression analysis of Arabidopsis exposed to RDX and AOC, which are considered significant environmental contaminants, and demonstrates the importance of microscopy methods for phytoremediation investigations.