Cellular and transcriptome signatures unveiled by single-cell RNA-Seq following ex vivo infection of murine splenocytes with Borrelia burgdorferi.

Introduction: Lyme disease, the most common tick-borne infectious disease in the US, is caused by a spirochetal pathogen Borrelia burgdorferi (Bb). Distinct host responses are observed in susceptible and resistant strains of inbred of mice following infection with Bb reflecting a subset of inflammatory responses observed in human Lyme disease. The advent of post-genomic methodologies and genomic data sets enables dissecting the host responses to advance therapeutic options for limiting the pathogen transmission and/or treatment of Lyme disease. Methods: In this study, we used single-cell RNA-Seq analysis in conjunction with mouse genomics exploiting GFP-expressing Bb to sort GFP+ splenocytes and GFP- bystander cells to uncover novel molecular and cellular signatures that contribute to early stages of immune responses against Bb. Results: These data decoded the heterogeneity of splenic neutrophils, macrophages, NK cells, B cells, and T cells in C3H/HeN mice in response to Bb infection. Increased mRNA abundance of apoptosis-related genes was observed in neutrophils and macrophages clustered from GFP+ splenocytes. Moreover, complement-mediated phagocytosis-related genes such as C1q and Ficolin were elevated in an inflammatory macrophage subset, suggesting upregulation of these genes during the interaction of macrophages with Bb-infected neutrophils. In addition, the role of DUSP1 in regulating the expression of Casp3 and pro-inflammatory cytokines Cxcl1, Cxcl2, Il1b, and Ccl5 in Bb-infected neutrophils were identified. Discussion: These findings serve as a growing catalog of cell phenotypes/biomarkers among murine splenocytes that can be exploited for limiting spirochetal burden to limit the transmission of the agent of Lyme disease to humans via reservoir hosts.

Borrelia peptidoglycan interacting Protein (BpiP) contributes to the fitness of Borrelia burgdorferi against host-derived factors and influences virulence in mouse models of Lyme disease.

The Peptidoglycan (PG) cell wall of the Lyme disease (LD) spirochete, Borrelia burgdorferi (Bb), contributes to structural and morphological integrity of Bb; is a persistent antigen in LD patients; and has a unique pentapeptide with L-Ornithine as the third amino acid that cross-links its glycan polymers. A borrelial homolog (BB_0167) interacted specifically with borrelilal PG via its peptidoglycan interacting motif (MHELSEKRARAIGNYL); was localized to the protoplasmic cylinder of Bb; and was designated as Borrelia peptidoglycan interacting Protein (BpiP). A bpiP mutant displayed no defect under in vitro growth conditions with similar levels of several virulence-related proteins. However, the burden of bpiP mutant in C3H/HeN mice at day 14, 28 and 62 post-infection was significantly lower compared to control strains. No viable bpiP mutant was re-isolated from any tissues at day 62 post-infection although bpiP mutant was able to colonize immunodeficient SCID at day 28 post-infection. Acquisition or transmission of bpiP mutant by Ixodes scapularis larvae or nymphs respectively, from and to mice, was significantly lower compared to control strains. Further analysis of bpiP mutant revealed increased sensitivity to vancomycin, osmotic stress, lysosomal extracts, human antimicrobial peptide cathelicidin-LL37, complement-dependent killing in the presence of day 14 post-infection mouse serum and increased internalization of CFSC-labeled bpiP mutant by macrophages and dendritic cells compared to control strains. These studies demonstrate the importance of accessory protein/s involved in sustaining integrity of PG and cell envelope during different phases of Bb infection.

Transformation of Borrelia burgdorferi.

Transformation techniques used to genetically manipulate Borrelia burgdorferi, the agent of Lyme disease, play a critical role in generating mutants that facilitate analyses of the role of genes in the pathophysiology of this bacterium. A number of borrelial mutants have been successfully isolated and characterized since the first electrotransformation procedure was established 25 years ago (Samuels, 1995). This article is directed at additional considerations for transforming infectious B. burgdorferi to generate strains retaining the plasmid profile of the parental strain, enabling analysis of transformants for in vitro and in vivo phenotypes. These methods are built on previously published protocols and are intended to add steps and tips to enhance transformation efficiency and recovery of strains amenable for studies involving colonization, survival, and transmission of B. burgdorferi during the vector and vertebrate phases of infection. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of stock cultures, propagation of spirochetes, and analysis of plasmid profiles Basic Protocol 2: Preparation of plasmid and linear DNA templates for transformation Basic Protocol 3: Transformation of B. burgdorferi Basic Protocol 4: Antibiotic selection of borrelial transformants Basic Protocol 5: Isolation of borrelial transformants in agar overlays Basic Protocol 6: Complementation of mutant borrelial strains in cis or in trans.

Cytotoxicity and genotoxicity of cadmium oxide nanoparticles evaluated using in vitro assays.

Cadmium oxide nanoparticles (CdO NPs) are among some of the most studied and industrially used metal oxide NPs. They have been widely used for industrial application, such as paint pigments and electronic devices, and medical therapeutics. With increasing use of CdO NPs and concerns for their potential adverse effects on the environment and public health, evaluation of the cytotoxicity and genotoxicity of CdO NPs becomes very important. To date, there is a limited understanding of the potential hazard brought by CdO NPs and a lack of information and research, particularly on the genotoxicity assessment of these NPs. In this study, 10 nm CdO core-PEG stabilized NPs were synthesized, characterized and used for evaluation of CdO NPs' cytotoxicity and genotoxicity. Release of cadmium ions (Cd+2) from the CdO NPs in cell culture medium, cellular uptake of the NPs, and the endotoxin content of the particles were measured prior to the toxicity assays. Cytotoxicity was evaluated using the MTS assay, ATP content detection assay, and LDH assay. Genotoxicity was assessed using the Ames test, Comet assay, micronucleus assay, and mouse lymphoma assay. The cytotoxicity of cadmium chloride (CdCl2) was also evaluated along with that of the CdO NPs. The results showed that endotoxin levels within the CdO NPs were below the limit of detection. CdO NPs induced concentration-dependent cytotoxicity in TK6 and HepG2 cells with the MTS, ATP and LDH assays. Although the genotoxicity of CdO NPs was negative in the Ames test, positive results were obtained with the micronucleus, Comet, and mouse lymphoma assays. The negative response of CdO NPs with the Ames test may be the result of unsuitability of the assay for measuring NPs, while the positive responses from other genotoxicity assays suggest that CdO NPs can induce chromosomal damage, single or double strand breaks in DNA, and mutations. The toxicity of the CdO NPs results from the NPs themselves and not from the released Cd+2, because the ions released from the NPs were minimal. These results demonstrate that CdO NPs are cytotoxic and genotoxic and provide new insights into risk assessment of CdO NPs for human exposure and environmental protection.

Study of pharmacogenomic information in FDA-approved drug labeling to facilitate application of precision medicine.

Pharmacogenomics (PGx), studying the relationship between drug response and genetic makeup of an individual, is accelerating advances in precision medicine. The FDA includes PGx information in the labeling of approved drugs to better inform on their safety and effectiveness. We herein present a summary of PGx information found in 261 prescription drug labeling documents by querying the publicly available FDALabel database. A total of 362 drug-biomarker pairs (DBPs) were identified. We profiled DBPs using frequency of the biomarkers and their therapeutic classes. Four categories of applications (indication, safety, dosing and information) were discussed according to information in labeling. This analysis facilitates better understanding, utilization and translation of PGx information in drug labeling among researchers, healthcare professionals and the public.

Study of serious adverse drug reactions using FDA-approved drug labeling and MedDRA.

BACKGROUND: Adverse Drug Reactions (ADRs) are of great public health concern. FDA-approved drug labeling summarizes ADRs of a drug product mainly in three sections, i.e., Boxed Warning (BW), Warnings and Precautions (WP), and Adverse Reactions (AR), where the severity of ADRs are intended to decrease in the order of BW > WP > AR. Several reported studies have extracted ADRs from labeling documents, but most, if not all, did not discriminate the severity of the ADRs by the different labeling sections. Such a practice could overstate or underestimate the impact of certain ADRs to the public health. In this study, we applied the Medical Dictionary for Regulatory Activities (MedDRA) to drug labeling and systematically analyzed and compared the ADRs from the three labeling sections with a specific emphasis on analyzing serious ADRs presented in BW, which is of most drug safety concern. RESULTS: This study investigated New Drug Application (NDA) labeling documents for 1164 single-ingredient drugs using Oracle Text search to extract MedDRA terms. We found that only a small portion of MedDRA Preferred Terms (PTs), 3819 out of 21,920 or 17.42%, were observed in a whole set of documents. In detail, 466/3819 (12.0%) PTs were in BW, 2023/3819 (53.0%) were in WP, and 2961/3819 (77.5%) were in AR sections. We also found a higher overlap of top 20 occurring BW PTs with WP sections compared to AR sections. Within the MedDRA System Organ Class levels, serious ADRs (sADRs) from BW were prevalent in Nervous System disorders and Vascular disorders. A Hierarchical Cluster Analysis (HCA) revealed that drugs within the same therapeutic category shared the same ADR patterns in BW (e.g., nervous system drug class is highly associated with drug abuse terms such as dependence, substance abuse, and respiratory depression). CONCLUSIONS: This study demonstrated that combining MedDRA standard terminologies with data mining techniques facilitated computer-aided ADR analysis of drug labeling. We also highlighted the importance of labeling sections that differ in seriousness and application in drug safety. Using sADRs primarily related to BW sections, we illustrated a prototype approach for computer-aided ADR monitoring and studies which can be applied to other public health documents.

Investigating the susceptibility of mice to a bacterial challenge after intravenous exposure to durable nanoparticles.

AIM: The goal of this study was to determine whether bacterial clearance in a rodent model would be impaired upon exposure to gold, silver or silica nanoparticles (NPs). MATERIALS & METHODS: Mice received weekly injections of NPs followed by a challenge of Listeria monocytogenes (LM). On days 3 and 10 after LM injections, the animals were sacrificed and their tissues were collected for elemental analysis, electron microscopy and LM count determination. RESULTS: The untreated and NP-treated animals cleared LM at the same rate suggesting that bioaccumulation of NPs did not increase the animals' susceptibility to bacterial infection. CONCLUSION: The data from this study indicate that the bioaccumulation of NPs does not significantly affect the ability to react to a bacterial challenge.

Evaluating the potential of gold, silver, and silica nanoparticles to saturate mononuclear phagocytic system tissues under repeat dosing conditions.

BACKGROUND: As nanoparticles (NPs) become more prevalent in the pharmaceutical industry, questions have arisen from both industry and regulatory stakeholders about the long term effects of these materials. This study was designed to evaluate whether gold (10 nm), silver (50 nm), or silica (10 nm) nanoparticles administered intravenously to mice for up to 8 weeks at doses known to be sub-toxic (non-toxic at single acute or repeat dosing levels) and clinically relevant could produce significant bioaccumulation in liver and spleen macrophages. RESULTS: Repeated dosing with gold, silver, and silica nanoparticles did not saturate bioaccumulation in liver or spleen macrophages. While no toxicity was observed with gold and silver nanoparticles throughout the 8 week experiment, some effects including histopathological and serum chemistry changes were observed with silica nanoparticles starting at week 3. No major changes in the splenocyte population were observed during the study for any of the nanoparticles tested. CONCLUSIONS: The clinical impact of these changes is unclear but suggests that the mononuclear phagocytic system is able to handle repeated doses of nanoparticles.

Differential genotoxicity mechanisms of silver nanoparticles and silver ions.

In spite of many reports on the toxicity of silver nanoparticles (AgNPs), the mechanisms underlying the toxicity are far from clear. A key question is whether the observed toxicity comes from the silver ions (Ag+) released from the AgNPs or from the nanoparticles themselves. In this study, we explored the genotoxicity and the genotoxicity mechanisms of Ag+ and AgNPs. Human TK6 cells were treated with 5 nM AgNPs or silver nitrate (AgNO3) to evaluate their genotoxicity and induction of oxidative stress. AgNPs and AgNO3 induced cytotoxicity and genotoxicity in a similar range of concentrations (1.00-1.75 µg/ml) when evaluated using the micronucleus assay, and both induced oxidative stress by measuring the gene expression and reactive oxygen species in the treated cells. Addition of N-acetylcysteine (NAC, an Ag+ chelator) to the treatments significantly decreased genotoxicity of Ag+, but not AgNPs, while addition of Trolox (a free radical scavenger) to the treatment efficiently decreased the genotoxicity of both agents. In addition, the Ag+ released from the highest concentration of AgNPs used for the treatment was measured. Only 0.5 % of the AgNPs were ionized in the culture medium and the released silver ions were neither cytotoxic nor genotoxic at this concentration. Further analysis using electron spin resonance demonstrated that AgNPs produced hydroxyl radicals directly, while AgNO3 did not. These results indicated that although both AgNPs and Ag+ can cause genotoxicity via oxidative stress, the mechanisms are different, and the nanoparticles, but not the released ions, mainly contribute to the genotoxicity of AgNPs.

Size- and coating-dependent cytotoxicity and genotoxicity of silver nanoparticles evaluated using in vitro standard assays.

The physicochemical characteristics of silver nanoparticles (AgNPs) may greatly alter their toxicological potential. To explore the effects of size and coating on the cytotoxicity and genotoxicity of AgNPs, six different types of AgNPs, having three different sizes and two different coatings, were investigated using the Ames test, mouse lymphoma assay (MLA) and in vitro micronucleus assay. The genotoxicities of silver acetate and silver nitrate were evaluated to compare the genotoxicity of nanosilver to that of ionic silver. The Ames test produced inconclusive results for all types of the silver materials due to the high toxicity of silver to the test bacteria and the lack of entry of the nanoparticles into the cells. Treatment of L5718Y cells with AgNPs and ionic silver resulted in concentration-dependent cytotoxicity, mutagenicity in the Tk gene and the induction of micronuclei from exposure to nearly every type of the silver materials. Treatment of TK6 cells with these silver materials also resulted in concentration-dependent cytotoxicity and significantly increased micronucleus frequency. With both the MLA and micronucleus assays, the smaller the AgNPs, the greater the cytotoxicity and genotoxicity. The coatings had less effect on the relative genotoxicity of AgNPs than the particle size. Loss of heterozygosity analysis of the induced Tk mutants indicated that the types of mutations induced by AgNPs were different from those of ionic silver. These results suggest that AgNPs induce cytotoxicity and genotoxicity in a size- and coating-dependent manner. Furthermore, while the MLA and in vitro micronucleus assay (in both types of cells) are useful to quantitatively measure the genotoxic potencies of AgNPs, the Ames test cannot.

Systemic and behavioral effects of intranasal administration of silver nanoparticles.

Use of silver nanoparticles (AgNPs) for their antimicrobial properties is widespread. Much of the previous work on the toxicity of AgNPs has been conducted in vitro or following oral or intravenous administration in vivo. Intranasal (IN) instillation of AgNPs mimics inhalation exposure and allows further exploration of the toxicity of these particles via respiratory tract exposure. The present study involved 1) single-dose exposures to assess tissue distribution and toxicity and 2) repeated exposures to assess behavioral effects of IN AgNP exposure (nominally uncoated 25 nm AgNP). AgNP deposition was localized in the liver, gut-associated lymphoid tissue, and brain. Decrease cellularity in spleen follicles was observed in treated mice, along with changes in cell number and populations in the spleen. The splenic GSH:GSSG ratio was also reduced following AgNP exposure. Expression of the oxidative stress-responsive gene Hmox1 was elevated in the hippocampus, but not cortex of treated mice, as was the level of HMOX1 protein. Mice receiving 7 days of IN exposure to 50 mg/kg AgNPs exhibited similar learning- and memory-related behaviors to control mice, except that treated mice spent significantly less time in the target quadrant of the Morris Water Maze during the acquisition phase probe trial. These findings indicate systemic distribution and toxicity following IN administration of AgNPs.

Rapid determination of plasmonic nanoparticle agglomeration status in blood.

Plasmonic nanomaterials as drug delivery or bio-imaging agents are typically introduced to biological systems through intravenous administration. However, the potential for agglomeration of nanoparticles in biological systems could dramatically affect their pharmacokinetic profile and toxic potential. Development of rapid screening methods to evaluate agglomeration is urgently needed to monitor the physical nature of nanoparticles as they are introduced into blood. Here, we establish novel methods using darkfield microscopy with hyperspectral detection (hsDFM), single particle inductively-coupled plasma mass spectrometry (spICP-MS), and confocal Raman microscopy (cRM) to discriminate gold nanoparticles (AuNPs) and their agglomerates in blood. Rich information about nanoparticle agglomeration in situ is provided by hsDFM monitoring of the plasmon resonance of primary nanoparticles and their agglomerates in whole blood; cRM is an effective complement to hsDFM to detect AuNP agglomerates in minimally manipulated samples. The AuNPs and the particle agglomerates were further distinguished in blood for the first time by quantification of particle mass using spICP-MS with excellent sensitivity and specificity. Furthermore, the agglomeration status of synthesized and commercial NPs incubated in blood was successfully assessed using the developed methods. Together, these complementary methods enable rapid determination of the agglomeration status of plasmonic nanomaterials in biological systems, specifically blood.

Effect of light, methyl jasmonate and cyclodextrin on production of phenolic compounds in hairy root cultures of Scutellaria lateriflora.

Scutellaria lateriflora (American skullcap) has been used in traditional medicine to treat several medical conditions including nervous disorders and cancer. Previous studies have associated these medicinal properties to flavones present in roots and leaves of this species. In order to develop a production system and study the biosynthesis of these bioactive compounds, hairy root cultures of S. lateriflora were established and line 4 was selected for further studies based on its growth performance in a modified Murashige and Skoog's medium supplemented with 0.5mg/l indole-3-butyric acid. Scanning electron microscopy of the hairy roots showed a high profusion of hairs along the root. Several phenolic compounds, including verbascoside, and the flavones wogonin, baicalein, scutellarein and their respective glucuronides were identified by high performance liquid chromatography-tandem mass spectrometry in the root tissue, but not in the culture medium. Among these compounds, verbascoside accumulated at the highest levels. Interestingly, cultures incubated under continuous light and treated with 15mM methyl-ß-cyclodextrin for 24h produced significantly higher levels of the aglycones, baicalein and wogonin, but not scutellarein, compared to cultures incubated under continuous darkness. This work demonstrates that hairy root cultures of S. lateriflora have the biosynthetic capacity to produce known Scutellaria flavones and suggest that light may have a selected regulatory effect on the synthesis or accumulation of these phenolic compounds.

Raman spectroscopy analysis and mapping the biodistribution of inhaled carbon nanotubes in the lungs and blood of mice.

Because of their small size, robust structure and unique characteristics, carbon nanotubes (CNTs) are increasingly being used in a variety of biomedical applications, materials and products. As their use increases, so does the probability of their unintended release and human exposure. Therefore, it is important to establish their potential biodistribution and biopersistence to better understand the potential effects of their exposure to humans. This study examines the distribution of CNTs in CD-1 mice after exposure by inhalation of single-walled carbon nanotubes (SWCNTs) and investigates the possibility that inhaled nanoparticles could enter the circulatory system via the lungs. Raman spectroscopy was employed for the detection of CNTs in lung tissue and blood based on their unique spectroscopic signatures. These studies have important implications concerning the potential effects of exposure to SWCNTs and their use as potential transport vehicles in nanomedicine.

Aqueous toxicity and food chain transfer of Quantum DOTs in freshwater algae and Ceriodaphnia dubia.

Innovative research and diagnostic techniques for biological testing have advanced during recent years because of the development of semiconductor nanocrystals. Although these commercially available, fluorescent nanocrystals have a protective organic coating, the inner core contains cadmium and selenium. Because these metals have the potential for detrimental environmental effects, concerns have been raised over our lack of understanding about the environmental fate of these products. U.S. Environmental Protection Agency test protocol and fluorescence microscopy were used to determine the fate and effect of quantum dots (QDs; Qdot 545 ITK Carboxyl Quantum Dots [Fisher Scientific, Fisher part Q21391MP; Invitrogen Molecular Probes, Eugene, OR, USA]) using standard aquatic test organisms. No lethality was measured following 48-h exposure of Ceriodaphnia dubia to QD suspensions as high as 110 ppb, but the 96-h median lethal concentration to Pseudokirchneriella subcapitata was measured at 37.1 ppb. Transfer of QDs from dosed algae to C. dubia was verified with fluorescence microscopy. These results indicate that coatings present on nanocrystals provide protection from metal toxicity during laboratory exposures but that the transfer of core metals from intact nanocrystals may occur at levels well above toxic threshold values, indicating the potential exposure of higher trophic levels. Studies regarding the fate and effects of nanoparticles can be incorporated into models for predictive toxicology of these emerging contaminants.

Absorption of semiconductor nanocrystals by the aquatic invertebrate Ceriodaphnia dubia.

When incubated with nanomolar concentrations of fluorescent semiconductor nanocrystals in moderately hard water the fluorescence of living Ceriodaphnia dubia increased. Average pixel intensity of exposed animals was greater than that of unexposed animals and increased in a dose and exposure time-dependent-manner. Internal structures were clearly visible in exposed and unexposed animals but fluorescence was most intense in the region of the abdominal appendages of exposed animals. Since these nanoparticles contain significant quantities of the toxic metals cadmium and selenium, their accumulation from aquatic environments by this standard test organism has significant implications about their potential environmental toxicity.