The microbiome has a profound impact on host fitness.pH, oxygen, nutrients, or other factors such as food or pharmaceuticals, subject the microbiome to variations in the gastrointestinal tract.This variation is a cause for concern given dysbiosis of the microbiome is correlated with various disease states.Currently, much research relies on model organisms to study microbial communities since intact microbiomes are challenging to utilize.The objective of this study is to culture an explanted colon microbiome of 4 Balb/c mice to develop an in vitro tool for future microbiome studies.We cultured homogenates of the distal colons of 4 mice in trans-well culture dishes.These dishes were incubated for 24 hours in two different oxygen concentration levels and the pH was compared before and after incubation of the cultures.To analyze the integrity of the microbiome, we utilized massively paralleled DNA sequencing with 16S metagenomics to characterize fecal and colon samples to speculate whether future studies may utilize feces in constructing an in vitro microbial community to spare animal lives.We found that pH and familial relationships had a profound impact on community structure while oxygen did not have a significant influence.The feces and the colon were similar in community profiles, which lends credence to utilizing feces in future studies.The gut microbiome is of great interest and great importance for studies in a variety of different diseases.Many laboratories do not have access to germ-free mice, which is one optimal way to study mammalian microbiomes, but this technique allowed for the in vitro culturing of a majority of the prokaryotes isolated from the colons of mice.This may allow an alternative to study the interactions of this very diverse population of microorganisms without the need for germ-free conditions.

Fluctuations in oxygen, pH, nutrients, or other factors such as food or pharmaceuticals, may perturb the microbiota of the gastrointestinal (GI) tract. This environmental variation is a cause for concern given dysbiosis of the microbiome is correlated with disease states; thereby, model organisms are utilized to study microbial communities during, after, or before shifts in microbes since intact ex vivo microbiomes have historically been challenging to utilize. The objective of this study is to culture an explant microbiome of 4 Balb/c, laboratory bred mice to develop an ex vivo tool for future microbiome studies. We cultured homogenates of the distal colon of 4 mice in three dimensional, 24 well plate culture dishes. These dishes were incubated for 24 hours in two different oxygen concentration levels, 0% and 20%. The pH of the plate was tested before and after incubation. To analyze the integrity of the microbiome, we utilized 16S sequencing. Further, we utilized 16S metagenomics to characterize fecal samples and colon samples to speculate whether future studies may utilize feces in constructing an explant microbiome to spare animal lives. We found that pH and familial relationship had a profound impact on community structure while oxygen did not have a significant influence. The feces and the colon were similar in community profiles, which lends credence to utilizing feces in future studies. In addition, our efforts successfully cultured archaea, which included difficult to culture strains such as Miscellaneous Crenarchaeota group (MCG) and Methanobacteria. Ultimately, further attempts to culture and preserve an animal’s microbiome needs to control for and maintain stable pH.

Respiratory syncytial virus (RSV) is an important etiological agent of respiratory infection in children for which no specific treatment option is available. The RSV virion contains two surface glycoproteins (F and G) that are vital for the initial phases of infection, making them critical targets for RSV therapeutics. Recent studies have identified the broad-spectrum antiviral properties of silver nanoparticles (AgNPs) against respiratory pathogens, such as adenovirus, parainfluenza, and influenza. AgNPs achieve this by attaching to viral glycoproteins, blocking entry into the host cell. The objective of this study was to evaluate the antiviral and immunomodulatory effects of AgNPs in RSV infection. Herein we demonstrate AgNP-mediated reduction in RSV replication, both in epithelial cell lines and in experimentally infected BALB/c mice. Marked reduction in pro-inflammatory cytokines (i.e., IL-1α, IL-6, TNF-α) and pro-inflammatory chemokines (i.e., CCL2, CCL3, CCL5) was also observed. Conversely, CXCL1, G-CSF, and GM-CSF were increased in RSV-infected mice treated with AgNPs, consistent with an increase of neutrophil recruitment and activation in the lung tissue. Following experimental antibody-dependent depletion of neutrophils, the antiviral effect of AgNPs in mice treated was ablated. To our knowledge, this is the first in vivo report demonstrating antiviral activity of AgNPs during RSV infection.

Samples are often frozen for preservation until needed for use. It has been a common practice to store fresh dairy manure in the freezer until needed for fly development studies. However, conflicting data have suggested that freezer temperature and duration of manure may impact fly development studies, and it is likely due to the change in microbial comminutes due to the freezer conditions. In this study manure storage conditions were assessed to ascertain how temperatures impact stable fly, Stomoxys calcitrans L., survival to pupation and determine which bacterial populations impacted fly development using massively-parallel sequencing and 16S metagenomic analysis. Stable fly survival to pupation was greater in manure that was stored warm (27˚C) or frozen (-20˚C or -80˚C) for 24 days as compared to fresh manure samples. Refrigeration (4˚C) of the manure for 24 days did not affect fly development and slightly decreased the pupal weights. Over 80 bacterial families were detected by sequencing allowing for a more thorough assessment of changes in bacterial populations. Only minor shifts were observed in bacterial family composition in the manure when refrigerated or frozen for 24 days, but significant population changes were observed when the manure was incubated for 24 days at 27˚C. Since it is the temperature and incubation time that yielded the greatest pupation rate, it is hypothesized that the manure microbial community impacts the growth and development of stable flies. This study has determined suggested freezer conditions for the best storage of manure samples to maintain bacterial diversity and retain the closest bacterial populations to freshly collected manure. Although untouched, aged (20 days) manure is best to use to assess fly development, it is not always feasible in laboratory experimentations. This study demonstrates the importance of preservation techniques on manure samples, which could also confer to storage of other biological specimens that contain resident microbes.

It has been well established that synthesis methods can cause variability in nanotoxicity studies, but here we demonstrate that environment can play a role as well. Silver nanoparticles were dispersed in two different environmentally-obtained fresh water samples for an ecotoxicological assessment of their impact on zebrafish. The two water sources varied in alkalinity, pH, and total hardness, and this variation impacted nanoparticle agglomeration size, uptake into the zebrafish kidneys, inflammation and apoptosis, and the zebrafish microbiota. *Correspondence to: Speshock J, Associate Professor, Department of Biological Sciences, Tarleton State University, 1333 W. Washington Street, Stephenville, TX 76401 USA, Tel: [254] 968-9341; E-mail: speshock@tarleton.edu

Streptococcus pneumoniae is a medically important pathogen capable of causing human infections of pneumonia, bacteremia, otitis media, and meningitis. Although there are vaccinations available, infections with S. pneumoniae still remains a global problem. S. pneumoniae is a highly adaptable bacterial species with numerous serotypes based on capsular polysaccharides. The different serotypes vary in their ability to colonize and causing pathology. Here we compared the regulation of five different virulence factors from four common serotypes of S. pneumoniae that vary in their carriage, morbidity, and mortality rates in the human population using two different in vitro methods, broth and cell culture. We determine that there is variation of virulence factor gene regulation within a serotype using two different culture methods, and variation between the serotypes in the same culture condition. The regulation of genes appeared to have a correlation with the ability of the various serotypes to grow in broth culture, adhere to cultured lung cells, and invade the cultured lung cells, as serotypes that shared similar regulation of virulence factors tended to behave similarly in culture. Many studies with S. pneumoniae rely on the use of one selected serotype, but since there is a wide variation in the growth and regulatory mechanisms of these bacteria. As demonstrated here, future studies should utilize more strains in models before concluding mechanisms of pathobiology.

Background: Arenaviruses are important pathogens that can cause hemorrhagic fever or meningoencephalitis. There is not an effective treatment targeting arenaviruses, which necessitates a search for therapies against this virus family, as well as others. Silver nanoparticles (Ag-NPs) have been shown to have effective antiviral activity in cell culture models against arenaviruses, and therefore it was hypothesized that they may make an effective therapeutic against viral meningitis. However, the silver nanoparticles interfered with normal cytokine profiles produced in response to the virus infection, which led to exacerbated pathology. Methods: Mice were infected with Tacaribe virus, a mouse-adapted arenavirus that causes lethal encephalitis. The virus was left untreated or treated with 10 nm Ag-NPs, which were demonstrated previously to inhibit virus replication in cell culture. Virus replication, brain pathology, and cytokine profiles (pro-inflammatory interleukins and type I interferons), as well as over morbidity and mortality were assessed in the mice with and without treatment. Results: Although the viral loads appeared to be reduced in the Ag-NP treated mice, mortality was observed in all virus-infected animals, regardless of whether or not they received nanoparticle therapy. The Ag-NP treatments also the innate immune response against the virus infection and unregulated the production of interleukin-1 beta, which likely contributed to the observed mortality in the mice. Conclusions: This study confirms why it is important to assess promising in vitro results using in vivo models. Biological barriers can impact the efficacy of therapeutics in mammals. Although Ag-NPs may still have success as an antiviral therapeutic in other areas of the body that are less constricted, in sensitive areas such as the brain, the nanoparticles can cause very adverse reactions.

Studies have suggested that silver nanoparticles can augment the cellular immune responses of cells and thus may contribute to immunopathology, especially as an autoinflammatory disorder.This study examines the interaction of silver nanoparticles with a co-culture of alveolar macrophages and lung epithelial cells to determine if the nanoparticles are capable of activating the inflammasome for sustained production of the pro-inflammatory cytokine interleukin-1beta.It is demonstrated here that interleukin-1beta is upregulated, both the transcript and the mature protein, following 24 hour exposure to 10 nm silver, but that caspase-1 function is downregulated, which prevents confirmation of the inflammasome activity.However, significant upregulation of interleukin-1beta is the contributing factor to many autoinflammatory disorders, indicating that prolonged exposure to silver nanoparticles, even at sub-lethal doses, could potentially cause pathological damage to the host through inflammation.

The novel anti-microbial properties of silver nanomaterials have made them desirable for use in health and medicine. However, prior to in vivo usage of any material, a thorough understanding of bio-distribution and potential toxicity is required. The majority of the toxicological data for silver nanoparticles (Ag-NPs) is still primarily from single-cell culture models, which can often be misleading and lack in vivo correlation. To develop a greater understanding of the fate and interactions of Ag-NPs within a living organism, we administered Ag-NPs to adult zebrafish via the intramuscular route, a common medical administration site, and tracked the fate and toxicity. Although at the doses used in the study there was no death, silver was detected in many of the major organs of the zebrafish, indicating that dispersion occurred from the site of inoculation. The heart, gall bladder, spleen, and kidney all demonstrated varying degrees of damage due to the nanoparticles, and further evaluation identified inflammation caused by the presence of silver as the cause, suggesting that toxicity may be a factor if Ag-NPs are used in a clinical setting.

N anoenergetic aluminum has potential military, medical, and industrial applications,1,5 yet very few studies have evaluated the risk associated with these materials. Currently, studies pertaining to the biological interactions of aluminum nanoparticles (Al NPs) are very limited. Previously, we demonstrated that Al2O3 NPs reduce cell viability in male germline stem cells in a concentrationdependent manner.6 In another study we identified that the surface coating alters the toxicity in rat alveolar macrophages.7 Currently, nanotoxicity studies performed using a wide range of nanomaterials in a variety of cellular models have demonstrated a dose-dependent effect, but one limitation from all of these studies is that realistic human exposure scenarios that describe what interactions occur following low levels of nanoparticle exposure have not been addressed. We cannot assume that just because the NPs are not killing a cell that the cell remains unaffected by their presence. Additionally, using unicellular in vitro models to represent complex tissues such as the lungs will not provide an exact depiction of how a multicellular tissue will respond, especially because many of the major organs within the body contain phagocytic cells to respond to localized damage (summarized in Table 1). However, in vitro cultures are useful for providing a preliminary foundation for studies to assess dosing ranges and probable mechanisms of toxicity and to allow the for the refinement of techniques before progressing to costly in vivo studies. Perhaps, a better in vitro scenario would be to produce cocultures of cells that include the immune cells that would be present to respond to the nanomaterials, providing an even more realistic in vitro scenario. The aim of this study was to examine the effects of Al NPs in an alveolar coculture model consisting of epithelial and immune cells, since ultrafine particles are found in pollution that can be easily inhaled and absorbed systemically and the majority of nanotoxicity studies have focused on lung exposure. Another objective of this study was to evaluate the changes *Address correspondence to saber.hussain@wpafb.af.mil.

Nanomaterials are being incorporated into many biological applications for use as therapeutics, sensors, or labels. Silver nanomaterials are being utilized for biological implants and wound dressings as an antiviral material, whereas gold nanomaterials are being used as biological labels or sensors due to their surface properties and biocompatibility. Cytotoxicity data of these materials are becoming more prevalent; however, little research has been performed to understand how the introduction of these materials into cells affects cellular processes. Here, we demonstrate the impact that silver and gold nanoparticles have on cathepsin activity in vitro. Cathepsins are important cellular proteases that are imperative for proper immune system function. We have selected to examine gold and silver nanoparticles due to the increased use of these materials in biological applications. This manuscript depicts how both of these types of nanomaterials affect cathepsin activity, which could impact the host's immune system and its ability to respond to pathogens. Cathepsin B activity decreases in a dose-dependent manner with all nanoparticles tested. Alternatively, the impact of nanoparticles on cathepsin L activity depends greatly on the type and size of the material.

On the basis of their uses in jet fuels and munitions, the most likely scenario for aluminum nanoparticle (NP) exposure is inhalation. NPs have been shown to be capable of penetrating deep into the alveolar regions of the lung, and therefore human alveolar macrophages (U937) with human type II pneumocytes (A549) were cultured together and exposed to NPs dispersed in an artificial lung surfactant to more accurately mimic the lung microenvironment. Two types of NPs were evaluated: aluminum (Al) and aluminum oxide (Al2O3). Following a 24-h incubation, cell viability was assessed using MTS, and mild toxicity was observed at higher doses with the U937 cells affected more than the A549. Since the U937 cells provided protection from NP toxicity, the cocultures were exposed to a benign concentration of NPs and infected with the respiratory pathogen community-associated methicillin-resistant Staphylococcus aureus (ca-MRSA) to determine any changes in cellular function. Phagocytosis assays demonstrated that the NPs impaired phagocytic function, and bacterial growth curves confirmed that this reduction in phagocytosis was not related to NP-bacteria interactions. Furthermore, NFkappaB PCR arrays and an IL-6 and TNF-alpha real time PCR demonstrated that both types of NPs altered immune response activation. This change was confirmed by ELISA assays that evaluated the secretion of IL-6, IL-8, IL-10, IL-1beta, and TNF-alpha and illustrated that the NPs repressed secretion of these cytokines. Therefore, although the NPs were not toxic to the cells, they did impair the cell's natural ability to respond to a respiratory pathogen regardless of NP composition.

Background Silver nanoparticles possess many unique properties that make them attractive for use in biological applications. Recently they received attention when it was shown that 10 nm silver nanoparticles were bactericidal, which is promising in light of the growing number of antibiotic resistant bacteria. An area that has been largely unexplored is the interaction of nanomaterials with viruses and the possible use of silver nanoparticles as an antiviral agent. Results This research focuses on evaluating the interaction of silver nanoparticles with a New World arenavirus, Tacaribe virus, to determine if they influence viral replication. Surprisingly exposing the virus to silver nanoparticles prior to infection actually facilitated virus uptake into the host cells, but the silver-treated virus had a significant reduction in viral RNA production and progeny virus release, which indicates that silver nanoparticles are capable of inhibiting arenavirus infection in vitro . The inhibition of viral replication must occur during early replication since although pre-infection treatment with silver nanoparticles is very effective, the post-infection addition of silver nanoparticles is only effective if administered within the first 2-4 hours of virus replication. Conclusions Silver nanoparticles are capable of inhibiting a prototype arenavirus at non-toxic concentrations and effectively inhibit arenavirus replication when administered prior to viral infection or early after initial virus exposure. This suggests that the mode of action of viral neutralization by silver nanoparticles occurs during the early phases of viral replication.