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Channel catfish virus disease (CCVD) is the principal viral disease in the United States catfish industry. The CCVD is caused by channel catfish virus (CCV), with mortality reaching up to 100% in fingerlings. CCV is assigned taxonomically to the family Alloherpesviridae, genus Ictalurivirus, species Ictalurid herpesvirus 1 (IcHV-1). To date, virulence, immunogenicity, and genome plasticity of the CCV field isolates have not been investigated. Three genotypes of CCV (IcHV-1A, IcHV-1B, and BCAHV) were identified using restriction fragment length polymorphism (RFLP) analysis. Virulence assessment of three representative isolates of RFLP groups suggests that IcHV-1B (pooled survival [mean ± SE]: 58.3% ± 2.6) showed significantly lower survival than IcHV-1A (68.6% ± 2.4). Re-exposure of the survivors with a representative of IcHV-1A and IcHV-1B isolates indicates a robust cross-protective effect (relative percent survival [RPS]: 80-100%). Antigenic determinants against anti-CCV monoclonal antibody Mab-95 were conserved among IcHV-1A, and IcHV-1B; however, BCAHV possesses antigenically distinct epitopes (Neutralization index [NI] = 0). Although BCAHV and CCV have nearly colinear genomes (except for the absence of ORF16A in CCV), they represent distinct species, given that nucleotide identity is 93.9%. Moreover, infectivity trials indicated that channel and hybrid catfish fingerlings might be refractive to LD50 (1.3×105 TCID50/L) dosage of BCAHV. However, previous exposure to BCAHV has protected the channel and hybrid catfish against the subsequent infection with the ATCC strain of CCV (RPS:100%). Next, two discriminatory duplex probe-based qPCR assays were designed and validated to diagnose latent IcHV-1A and IcHV-1B. Spatio-temporal survey of six Mississippi catfish hatcheries indicated that the prevalence of latent CCV genotypes varied between 25-100%. Lastly, twenty one reference quality genomes of CCV field isolates were assembled, and phylogenomic analyses supported the monophyly of the CCV field isolates with BCAHV as their closest relative. The phylogenomic analyses confirmed the two distinct genotypes (IcHV-1A and IcHV-1B) identified in RFLP analysis and further allowed the segregation of the IcHV-1A genotype into two subgroups, IcHV-1A1 and IcHV-1A2. Results from the current studies lay the foundation for future research and will help formulate efficient management strategies to reduce the economic impact of CCV in the catfish industry.

Oral health is a part of general health and the two are connected in many ways as well as impact each other. Oral diseases are some of the most common chronic diseases of mankind. Diseases such as caries and periodontitis are two of the most common ones affecting the oral cavity. Bacteria associated with these two diseases are Streptococcus mutans and Aggregatibacter actinomycetemcomitans respectively. Our hypothesis is that extracts from guava leaf or matcha tea affect adhesion of A. actinomycetecomitans or S. mutans to human epithelium. The aim of this study is to investigate a DNA-based method for studying attachment of bacteria to epithelial cells. Two different concentrations of the two bacterial species, high and low, were treated with matcha or guava leaf extract and adhesion on the human epithelial cell cultures was analyzed. The results were then analyzed using qPCR-based methods to test the amount of bacterial adhesion to the epithelial cells. Furthermore, the results showed that matcha was more effective for inhibition of bacterial adhesion than guava leaf.In conclusion, our results show that bacterial adhesion of A. actinomycetecomitans and S. mutans to human epithelial cells can be quantified by DNA-based methods, and the adhesion altered by plant extracts.

Skin infections by keratinophilic fungi are commonly referred to as dermatophytosis and represent a major health burden worldwide. Although patient numbers are on the rise, data on virulence factors, their function and kinetics are scarce. We employed an ex vivo infection model based on guinea pig skin explants (GPSE) for the zoonotic dermatophyte Trichophyton (T.) benhamiae to investigate kinetics of the virulence factors subtilisin (sub) 3, sub 6, metallocarboxypeptidase A (mcpA) and isocitrate lyase (isol) at gene level for ten days. Fluorescence in situ hybridization (FISH) and quantitative polymerase chain reaction (qPCR) were used to detect and quantify the transcripts, respectively. Kingdom-spanning, species-specific and virulence factor-specific probes were successfully applied to isolated fungal elements showing inhomogeneous fluorescence signals along hyphae. Staining results for inoculated GPSE remained inconsistent despite thorough optimization. qPCR revealed a significant increase of sub 3- and mcpA-transcripts toward the end of culture, sub 6 and isol remained at a low level throughout the entire culture period. Sub 3 is tightly connected to the de novo formation of conidia during culture. Since sub 6 is considered an in vivo disease marker. However, the presented findings urgently call for further research on the role of certain virulence factors during infection and disease.

Previously, we described prostate cancer (PCa) detection (83% sensitivity; 67% specificity) in seminal plasma by CE-MS/MS. Moreover, advanced disease was distinguished from organ-confined tumors with 80% sensitivity and 82% specificity. The discovered biomarkers were naturally occurring fragments of larger seminal proteins, predominantly semenogelin 1 and 2, representing endpoints of the ejaculate liquefaction. Here we identified proteases putatively involved in PCa specific protein cleavage, and examined gene expression and tissue protein levels, jointly with cell localization in normal prostate (nP), benign prostate hyperplasia (BPH), seminal vesicles and PCa using qPCR, Western blotting and confocal laser scanning microscopy. We found differential gene expression of chymase (CMA1), matrix metalloproteinases (MMP3, MMP7), and upregulation of MMP14 and tissue inhibitors (TIMP1 and TIMP2) in BPH. In contrast tissue protein levels of MMP14 were downregulated in PCa. MMP3/TIMP1 and MMP7/TIMP1 ratios were decreased in BPH. In seminal vesicles, we found low-level expression of most proteases and, interestingly, we also detected TIMP1 and low levels of TIMP2. We conclude that MMP3 and MMP7 activity is different in PCa compared to BPH due to fine regulation by their inhibitor TIMP1. Our findings support the concept of seminal plasma biomarkers as non-invasive tool for PCa detection and risk stratification.

En 10-plex GeneXpert analysmetod för diagnos av M. Tuberculosis infektion samt med kapacitet att särskilja aktiv och latent tuberkulos har utvecklats på Cepheid. Analysmetoden sker ex-vivo och kvantifierar uttryck av tuberkulos associerade mRNA biomarkörer i antigenstimulerat helblod. Ett original set av mRNA biomarkörer har nedselekterats till 9 biomarkörer som utgör mRNA signaturen i 10-plex analysmetoden. Urvalet av biomarkörer baserades på probescreening och optimeringstudier utfört med Cepheids ”in cartridge” qPCR teknologi. Den diagnostiska prestandan av mRNA signaturen utvärderades genom att analysera 380 antigenstimulerade kliniska prover klassificerade som M. Tuberculosis infekterad, aktiv tuberkulos, latent tuberkulos och icke infekterad. mRNA signaturens förmåga korrekt klassificera samt diskriminera aktiv och latent tuberkulos bestämdes genom att implementera ROC-kurvor vilket resulterade i ett optimalt AUC-värde på 0.784 vilket visar på diagnostisk kapacitet att separera aktiv och latent tuberkulos. / A 10-color prototype GeneXpert assay based on distinct mRNA expression profiles in ex-vivo antigen stimulated whole blood has been developed at Cepheid for diagnosis of M. Tuberculosis infection and separation of active and latent tuberculosis. An original larger set of mRNA biomarkers has been downselected to a set of 9 biomarkers constituting the mRNA signature in the 10-color assay. The downselection of biomarkers was based on probe screening and optimization studies performed with Cepheid’s “in cartridge” qPCR technology. The 10-color assay was evaluated for diagnostic performance by analyzing 380 banked antigen stimulated clinical samples classified as M. Tuberculosis infected, active tuberculosis, latent tuberculosis and not infected. The ability of the mRNA signature to correctly classify and separate active and latent tuberculosis was determined by implementing ROC curves using delta Ctvalues as test variables and resulted in an optimal AUC value of 0.784 indicating ability to separate active and latent tuberculosis.

For several decades amphibian populations have been declining. Historically, the bacterium A. hydrophila (Ah) was hypothesized to be the causal factor in amphibian disease and population declines. However, with the discovery of a chytrid fungus, Batrachochytrium dendrobatidis (Bd) in 1998, which was identified on the skin of amphibians during documented mortality events, Ah research became of minor interest as focus shifted to Bd. Recent studies into the immunocompromising abilities of Bd, however, have opened new questions about its relationship with Ah and their combined effects on a host. In this study, I explore the relationship between infection with these two pathogens, Bd and Ah, in two amphibian species from distinct regions of the United States. I developed a novel qPCR assay to measure the microbial load of Ah on the skin of two anuran species, Lithobates yavapaiensis (N=232) and Pseudacris ornata (N=169), which have confirmed Bd infections. I use a logistic regression model to identify whether significant relationships exist between these two pathogens, disease, and death. I find that even amongst the most severely infected frogs, Ah is not detectable on the skin and only appears post-mortem. I therefore conclude that Ah is an opportunistic bacterial pathogen, scavenging on anurans only after mortality events. This research is the first known study to quantitatively assess Ah in amphibians in conjunction with Bd. While there is no causal relationship between these pathogens, future work will examine potential Ah infections in other organs to more fully understand the relationship between Bd and Ah.

For the past few decades, the popularity of graphene oxide (GO) nanomaterials (NMs) has increased exceedingly due to their biomedical applications in drug delivery of anti-cancer drugs. Their unique physicochemical properties such as high surface area and good surface chemistry with unbound surface functional groups (e.g. hydroxyl - OH, carboxyl /ketone C=O, epoxy/alkoxy C-O, aromatic group C=C, etc) which enable covalent bonding with organic molecules (e.g. RNA, DNA) make GO NMs as excellent candidates in drug delivery nanocarriers. Despite the overwhelming biomedical applications, there are concerns about their genotoxicity on human DNA. Published genotoxicity studies on GO NMs were performed using non-commercial GO with 2-3 layers of GO sheets, synthesized in various laboratories with the potential for inter-laboratory variabilities. However, what has not been studied before is the effects of the commercial GO (15-20 sheets; 4-10% edge-oxidized; 1 mg/mL) in vitro on DNA damage in human whole blood and peripheral blood lymphocytes (PBL) from real-life patients diagnosed with chronic pulmonary diseases [asthma, chronic obstructive pulmonary disease (COPD), and lung cancer], and genotoxic endpoints compared with those from healthy control individuals to determine whether there are any differences in GO sensitivity. Thus, in the present study, we had characterized GO NMs using Zetasizer Nano for Dynamic Light Scattering (DLS) and zeta potential (ZP) in the aqueous solution, and electron microscopy using the Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) in the dry state, respectively. Cytotoxicity studies were conducted on human PBL from healthy individuals and patients (asthma, COPD, and lung cancer) using the Methylthiazolyldiphenyl-tetrazolium bromide (MTT) and Neutral Red Uptake (NRU) assays, respectively. The genotoxicity (DNA damage) and cytogenetic effects (chromosome aberration parameters) induced by GO NMs on human whole blood from healthy individuals and patients were studied using the Alkaline Comet Assay and Cytokinesis-blocked Micronucleus (CBMN) assay, respectively. Our results showed concentration-dependent increases in cytotoxicity, genotoxicity, and chromosome aberrations, with blood samples from COPD and lung cancer patients being more sensitive to DNA damage insults compared with asthma patients and healthy control individuals. Furthermore, the relative gene and protein expressions of TP53, CDKN1A/p21, and BCL-2 relative to GAPDH on human PBL were studied using the Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) and Western Blot techniques, respectively. Our results have shown altered gene and protein expression levels. Specifically, GO-induced cytotoxicity, genotoxicity, and micronuclei aberrations were associated with TP53 upregulation - a biomarker of DNA damage - in both patients and healthy individuals. These effects show that GO NMs have promising roles in drug delivery applications when formulated to deliver drug payload to COPD and cancer cells. However, the fact that cytotoxicity, genotoxicity, chromosome instability, and gene/protein expressions - biomarkers of cancer risk - were observed in healthy individuals are of concern to public health, especially in occupational exposures at micro levels at the workplace.

No description available.

No description available.