Multiple Body Fluids Research Articles

LC-MS/MS quantification of felinine metabolites in tissues, fluids, and excretions from the domestic cat (Felis catus).

Domestic cat urine contains large concentrations of the unusual amino acid, felinine (2-amino-7-hydroxy-5,5-dimethyl-4-thiaheptanoic acid). A felinine derivative, 3-mercapt-3-methylbutanol is a potential scent signal for letting other animals know that the scent owners are cats. 3-Methylbutanol-glutathione (MBG) is an upstream precursor of the biosynthetic pathway of felinine that may be produced in hepatocytes by conjugation of glutathione with isopentenyl pyrophosphate, an intermediate for cholesterol biosynthesis. However, little evidence exists to support the biosynthesis of MBG in the liver. This study examined the distribution of metabolites of the felinine biosynthetic pathway in multiple tissues, body fluids, and excretions of cats. MBG, the felinine precursor, 3-methylbutanol-cysteinylglycine (MBCG), felinine, and felinine N-acetyl derivative were quantified by liquid chromatography-electron spray ionization-tandem mass spectrometry. All compounds were detected in cat serum. In males, MBG and MBCG contents were significantly higher than felinine and N-acetylfelinine contents. MBG was detected in multiple tissues, including the salivary gland, heart, liver, spleen, gut, kidney, bladder, adipose tissue, and muscle. Sex differences in MBG levels were observed in the liver and other tissues. Felinine and N-acetylfelinine were also detected in those tissues. Furthermore, we detected all compounds in cat bile and fecal samples, indicating that felinine is excreted into the feces via bile from the liver. We conclude that MBG is synthesized in several tissues in a sex-dependent manner. These findings improve our understanding of felinine metabolism and function in cats.

Exosomal miRNAs in nipple aspirate fluid and breast cancer

Background: Exosomal vesicles transport genetic information between cells and thereby influence the cells they interact with. Exosomes have been detected in multiple body fluids, including blood, milk, urine, pleural fluid, cerebrospinal fluid and the aqueous humor of the eye. We performed a proof of principle study to determine: (I) if exosomes are detectable in breast nipple aspirate fluid (NAF); (II) if exosomes are present, do they contain micro (mi)RNAs; (III) if present and if they contain miRNAs, the ability of the miRNAs to predict: (i) breast cancer; and (ii) response to treatment with the anti-inflammatory/anti-cancer agents vitamin (vit) D and celecoxib. Methods: NAF was collected from 12 healthy women and 20 women with newly diagnosed breast cancer. NAF and matched blood were collected from 5 women before and after daily treatment for one menstrual cycle/one month with low (400 IU) or high dose (2,000 IU) vitD, or high dose vitD plus 400 mg celecoxib. Ten miRs (16, 21, 100, 129, 145, 155, 181, 199, 205, and 212) were measured in NAF and serum samples. Results: We were routinely able to detect exosomes in NAF and serum samples, and in the exosomes to measure multiple miRNAs. Although there was no significant difference in expression based on the presence or absence of breast cancer, miR16 and -155 expressions were higher (P=0.030 for each) in women with node positive compared to those with node negative breast cancer. ∆Ct expression was at higher (defined at ∆Ct difference ≥1) in serum than matched NAF in healthy women. Conclusions: Exosomes are detectable in NAF. The exosomes contain intracellular information that may be useful for evaluation of the breast.

Phenotype-driven identification of modules in a hierarchical map of multifluid metabolic correlations

The identification of phenotype-driven network modules in complex, multifluid metabolomics data poses a considerable challenge for statistical analysis and result interpretation. This is the case for phenotypes with only few associations ('sparse' effects), but, in particular, for phenotypes with a large number of metabolite associations ('dense' effects). Herein, we postulate that examining the data at different layers of resolution, from metabolites to pathways, will facilitate the interpretation of modules for both the sparse and the dense cases. We propose an approach for the phenotype-driven identification of modules on multifluid networks based on untargeted metabolomics data of plasma, urine, and saliva samples from the German Study of Health in Pomerania (SHIP-TREND) study. We generated a hierarchical, multifluid map of metabolism covering both metabolite and pathway associations using Gaussian graphical models. First, this map facilitates a fundamental understanding of metabolism within and across fluids for our study, and can serve as a valuable and downloadable resource. Second, based on this map, we then present an algorithm to identify regulated modules that associate with factors such as gender and insulin-like growth factor I (IGF-I) as examples of traits with dense and sparse associations, respectively. We found IGF-I to associate at the rather fine-grained metabolite level, while gender shows well-interpretable associations at pathway level. Our results confirm that a holistic and interpretable view of metabolic changes associated with a phenotype can only be obtained if different layers of metabolic resolution from multiple body fluids are considered.

Implementation of a study to examine the persistence of Ebola virus in the body fluids of Ebola virus disease survivors in Sierra Leone: Methodology and lessons learned.

BackgroundThe 2013–2016 West African Ebola virus disease epidemic was unprecedented in terms of the number of cases and survivors. Prior to this epidemic there was limited data available on the persistence of Ebola virus in survivors’ body fluids and the potential risk of transmission, including sexual transmission.Methodology/Principal findingsGiven the urgent need to determine the persistence of Ebola virus in survivors’ body fluids, an observational cohort study was designed and implemented during the epidemic response operation in Sierra Leone. This publication describes study implementation methodology and the key lessons learned. Challenges encountered during implementation included unforeseen duration of follow-up, complexity of interpreting and communicating laboratory results to survivors, and the urgency of translating research findings into public health practice. Strong community engagement helped rapidly implement the study during the epidemic. The study was conducted in two phases. The first phase was initiated within five months of initial protocol discussions and assessed persistence of Ebola virus in semen of 100 adult men. The second phase assessed the persistence of virus in multiple body fluids (semen or vaginal fluid, menstrual blood, breast milk, and urine, rectal fluid, sweat, saliva, tears), of 120 men and 120 women.Conclusion/SignificanceData from this study informed national and global guidelines in real time and demonstrated the need to implement semen testing programs among Ebola virus disease survivors. The lessons learned and study tools developed accelerated the implementation of such programs in Ebola virus disease affected countries, and also informed studies examining persistence of Zika virus. Research is a vital component of the public health response to an epidemic of a poorly characterized disease. Adequate resources should be rapidly made available to answer critical research questions, in order to better inform response efforts.

High resolution melt curve analysis based on methylation status for human semen identification.

A high resolution melt curve assay to differentiate semen from blood, saliva, urine, and vaginal fluid based on methylation status at the Dapper Isoform 1 (DACT1) gene was developed. Stains made from blood, saliva, urine, semen, and vaginal fluid were obtained from volunteers and DNA was isolated using either organic extraction (saliva, urine, and vaginal fluid) or Chelex® 100 extraction (blood and semen). Extracts were then subjected to bisulfite modification in order to convert unmethylated cytosines to uracil, consequently creating sequences whose amplicons have melt curves that vary depending on their initial methylation status. When primers designed to amplify the promoter region of the DACT1 gene were used, DNA from semen samples was distinguishable from other fluids by a having a statistically significant lower melting temperature. The assay was found to be sperm-significant since semen from a vasectomized man produced a melting temperature similar to the non-semen body fluids. Blood and semen stains stored up to 5months and tested at various intervals showed little variation in melt temperature indicating the methylation status was stable during the course of the study. The assay is a more viable method for forensic science practice than most molecular-based methods for body fluid stain identification since it is time efficient and utilizes instrumentation common to forensic biology laboratories. In addition, the assay is advantageous over traditional presumptive chemical methods for body fluid identification since results are confirmatory and the assay offers the possibility of multiplexing which may test for multiple body fluids simultaneously.

Circulating MicroRNAs as Biomarkers in Biliary Tract Cancers.

Biliary tract cancers (BTCs) are a group of highly aggressive malignant tumors with a poor prognosis. The current diagnosis is based mainly on imaging and intraoperative exploration due to brush cytology havinga low sensitivity and the standard markers, such as carcinoembryonic antigen (CEA) and carbohydrate 19-9 (CA19-9), not having enough sensitivity nor specificity to be used in a differential diagnosis and early stage detection. Thus, better non-invasive methods that can distinguish between normal and pathological tissue are needed. MicroRNAs (miRNAs) are small, single-stranded non-coding RNA molecules of ~20–22 nucleotides that regulate relevant physiological mechanisms and can also be involved in carcinogenesis. Recent studies have demonstrated that miRNAs are detectable in multiple body fluids, showing great stability, either free or trapped in circulating microvesicles, such as exosomes. miRNAs are ideal biomarkers that may be used in screening and prognosis in biliary tract cancers, aiding also in the clinical decisions at different stages of cancer treatment. This review highlights the progress in the analysis of circulating miRNAs in serum, plasma and bile as potential diagnostic and prognostic markers of BTCs.

Human mast cells release extracellular vesicle-associated DNA.

Extracellular vesicles (EVs) carry multiple bioactive molecules, including proteins and nucleic acids. Cell-free, extracellular DNA has been reported to be present in cell cultures and in multiple body fluids, but its relative location in relation to EVs has not been described. This study demonstrates that DNase sensitive nucleic acids are present on the surface of EV isolates. Association of EV-DNA was revealed by increase of EV zeta potential and particle number upon DNase treatment. Additionally, Cells exposed to EVs with associated DNA, show the presence of cytoplasmic DNA traces intracellularly. In conclusion, we suggest that DNA can be associated to the surface of EVs, and can be taken up by recipient cells. DNA on EV surfaces may influence their function in recipient cells.

Interpretation of Negative Molecular Test Results in Patients With Suspected or Confirmed Ebola Virus Disease: Report of Two Cases.

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is the most sensitive quantitative diagnostic assay for detection of Ebola virus in multiple body fluids. Despite the strengths of this assay, we present 2 cases of Ebola virus disease (EVD) and highlight the potential for false-negative results during the early and late stages of EVD. The first case emphasizes the low negative-predictive value of qRT-PCR during incubation and the early febrile stage of EVD, and the second case emphasizes the potential for false-negative results during recovery and late neurologic complications of EVD. Careful interpretation of test results are needed to guide difficult admission and discharge decisions in suspected or confirmed EVD.

Network-based approach for analyzing intra- and interfluid metabolite associations in human blood, urine, and saliva.

Most studies investigating human metabolomics measurements are limited to a single biofluid, most often blood or urine. An organism's biochemical pool, however, comprises complex transboundary relationships, which can only be understood by investigating metabolic interactions and physiological processes spanning multiple parts of the human body. Therefore, we here propose a data-driven network-based approach to generate an integrated picture of metabolomics associations over multiple fluids. We performed an analysis of 2251 metabolites measured in plasma, urine, and saliva, from 374 participants of the Qatar Metabolomics Study on Diabetes (QMDiab). Gaussian graphical models (GGMs) were used to estimate metabolite-metabolite interactions on different subsets of the data set. First, we compared similarities and differences of the metabolome and the association networks between the three fluids. Second, we investigated the cross-talk between the fluids by analyzing correlations occurring between them. Third, we propose a framework for the analysis of medically relevant phenotypes by integrating type 2 diabetes, sex, age, and body mass index into our networks. In conclusion, we present a generic, data-driven network-based approach for structuring and visualizing metabolite correlations within and between multiple body fluids, enabling unbiased interpretation of metabolomics multifluid data.

Considering the effect of stem-loop reverse transcription and real-time PCR analysis of blood and saliva specific microRNA markers upon mixed body fluid stains

Forensic RNA analysis is gathering pace with reports of messenger RNA analysis being used in case work, and with microRNA being increasingly researched. Such techniques address a fundamental issue in body fluid identification, namely increased specificity over existing chemical tests, and the incorporation of additional body fluids such as vaginal material. The use of RNA analysis will be of particular value to sex offences, where there can be a mixture of multiple body fluids from different people. The aim of this study was to determine whether microRNA based body fluid identification tests can be applied to mixed body fluid samples. Blood and saliva were acquired from volunteers and underwent total RNA extraction. Mixed samples were prepared using a range of ratios from 1:1 to 10:1. Each mixed sample then underwent a blood-saliva differentiation test developed in-house, which includes stem-loop reverse transcription and real-time PCR analysis. Aliquots following mixture preparation also underwent standard STR analysis, utilising Quantiplex and Next Generation Multiplex kits. Data relating to the development of an in-house blood-saliva differentiation test is presented, in which it has been demonstrated that such a test has a lower limit of detection than the enzymatic equivalent. It has been shown that not only is it possible to determine the presence of more than one body fluid, it is also possible to determine the major body fluid contributor as well as the minor contributor.

Cancer biomarker discovery in saliva by mass spectrometry

The quest for biomarkers has been much pursued to aid in the early diagnosis, monitor post-treatment progress and development of targeted therapies. Nevertheless, the translation of biomarker discovery to clinical use has been limited due to multiple reasons such as the long path from discovery to clinical assays, limitation of samples and incoherent pipeline for biomarker development. To date, diagnosis of cancer has been based on biopsies and histological examinations and often becomes difficult to get repeated sampling from patients for confirmation. Consequently, it is important for clinical researchers to look at multiple body fluids and different molecular techniques to identify biomarkers. One such bodyfluid is saliva, which is easily and non-invasively collected and contains thousands of potential protein biomarkers. Moreover, recent advances in the sensitivity and specificity of mass spectrometry based proteomics hold great promise to identify potential biomarkers. This review presents an overview of the potential use of saliva and mass spectrometry for global discovery and validation of biomarkers.

Trash or Treasure: extracellular microRNAs and cell-to-cell communication

Circulating RNAs in human body fluids are promising candidates for diagnostic purposes. However, the biological significance of circulating RNAs remains elusive. Recently, small non-coding RNAs, microRNAs (miRNAs), were isolated from multiple human body fluids, and these “circulating miRNAs” have been implicated as novel disease biomarkers. Concurrently, miRNAs were also identified in the extracellular space associated with extracellular vesicles (EVs), which are small membrane vesicles secreted from various types of cells. The function of these secreted miRNAs has been revealed in several papers. Circulating miRNAs have been experimentally found to be associated with EVs; however, other types of extracellular miRNAs were also described. This review discusses studies related to extracellular miRNAs, including circulating miRNAs and secreted miRNAs, to highlight the importance of studying not only secreted miRNAs, but also circulating miRNAs to determine the contribution of extracellular miRNAs especially in cancer development.

Pulmonary Histoplasmosis

Pulmonary histoplasmosis is an important cause of morbidity in the United States. Several outbreaks of acute pulmonary histoplasmosis have been linked to potentially preventable environmental exposures. Progressive disseminated histoplasmosis, which is seen frequently in the growing population of immunocompromised hosts, often presents with prominent pulmonary manifestations and is more commonly encountered in hospitalized patients than acute, subacute, or chronic pulmonary histoplasmosis. A battery of diagnostic studies including serology, antigen, cytology/histopathology, and culture should be obtained in suspected cases of histoplasmosis. The yield of antigenuria detection is highest when the multiple body fluids are tested; the level of antigenuria correlates with severity of disease. Amphotericin B is the treatment of choice for severe pulmonary or disseminated histoplasmosis, and itraconazole is effective for mild to moderately severe infection. Posaconazole exhibits promise as a salvage agent. Antifungal prophylaxis is not routinely recommended for at-risk populations. Measures to minimize environmental contamination may reduce the risk of epidemic-type acute pulmonary histoplasmosis related to high-risk exposures.

Identification of forensically relevant body fluids using a panel of differentially expressed microRNAs

The serology-based methods routinely used in forensic casework for the identification of biological fluids are costly in terms of time and sample and have varying degrees of sensitivity and specificity. Recently, the use of a molecular genetics-based approach using messenger RNA (mRNA) profiling has been proposed to supplant conventional methods for body fluid identification. However, the size of the amplification products used in these mRNA assays (∼ 200–300 nt) might not be ideal for use with degraded or compromised samples frequently encountered in forensic casework. Recently, there has been an explosion of interest in a novel class of small noncoding RNAs, microRNAs (miRNAs, ∼20–25 bases in length), with numerous published studies reporting that some miRNAs are expressed in a tissue-specific manner. In this article, we provide the first comprehensive evaluation of miRNA expression in dried, forensically relevant biological fluids—blood, semen, saliva, vaginal secretions, and menstrual blood—in an attempt to identify putative body fluid-specific miRNAs. Most of the 452 human miRNAs tested (∼67% of the known miRNA ome) were either expressed in multiple body fluids or not expressed at all. Nevertheless, we have identified a panel of nine miRNAs—miR451, miR16, miR135b, miR10b, miR658, miR205, miR124a, miR372, and miR412—that are differentially expressed to such a degree as to permit the identification of the body fluid origin of forensic biological stains using as little as 50 pg of total RNA. The miRNA-based body fluid identification assays were highly specific because the miRNA expression profile for each body fluid was different from that obtained from 21 human tissues. The results of this study provide an initial indication that miRNA profiling may provide a promising alternative approach to body fluid identification for forensic casework.

Risk factors for human Herpesvirus 8 infection and AIDS‐associated Kaposi's sarcoma among men who have sex with men in a European multicentre study

We aimed to identify risk factors for Kaposi's sarcoma (KS) among HIV-positive patients and behaviors associated with human Herpesvirus 8 (HHV-8) infection, as well as to assess KS incidence and mortality rates longitudinally. To fulfill the first objective, a European case-control study was designed in the early 1990s (each KS case was matched to 2 controls with another AIDS indicative disease). After the discovery of HHV-8, serology testing enabled us to assess risk factors for KS development among HHV-8 and HIV-1 coinfected men who have sex with men (MSM), as well as risk factors for HHV-8 infection. HHV-8 seroprevalence was determined using a latent immunofluorescence assay. Relevant information was obtained by means of a questionnaire and medical charts review. Assessment of risk factors for KS development and HHV-8 infection was performed using conditional and unconditional logistic regression models, respectively. A low CD4 count was the only significant risk factor for KS. HHV-8 infection was most strongly linked to the number of life-time sex partners, and multiple body fluids such as saliva and semen are quite likely involved in sexual transmission. Longitudinal follow up showed a significant protective role for highly-active antiretroviral therapy (HAART) both on KS development and mortality of KS patients. Although more conclusive data from cohort studies are needed to better define specific transmission mechanisms for HHV-8, our results contribute to explain why KS incidence is higher among MSM, and the decreasing KS incidence trend observed in countries with universal access to HAART.

Genetic diversity among Mycoplasma species bovine group 7: clonal isolates from an outbreak of polyarthritis, mastitis, and abortion in dairy cattle.

A comprehensive genetic analysis of 60 Mycoplasma sp. bovine group 7 isolates from different geographic origins and epidemiological settings is presented. Twenty-four isolates were recovered from the joints of calves during sporadic episodes of polyarthritis in geographically distinct regions of Queensland and New South Wales, Australia, including two clones of the type strain PG5O. A further three Australian isolates were also recovered from the tympanic bulla, retropharyngeal lymph node and the lung and another three isolates had unconfirmed histories. Six isolates originated from Germany, Portugal, Nigeria, and France. Twenty-four epidemiologically related isolates of Mycoplasma sp. bovine group 7 were recovered from multiple tissue sites and body fluids of infected calves with polyarthritis, mastitic milk, and from the stomach contents, lung and liver from aborted foetuses in three large, centrally managed dairy herds in New South Wales, Australia. Restriction endonuclease analysis (REA) of genomic DNA differentiated 29 Cfol profiles among these 60 isolates and grouped all 24 epidemiologically related isolates in a defined pattern showing a clonal origin. Three isolates of this clonal cluster were recovered from mastitic milk and the synovial exudate of clinically-affected calves and appeared sporadically for periods up to 18 months after the initial outbreak of polyarthritis indicating a persistent, close association of the organism with cattle in these herds. The Cfol profile representative of the clonal cluster was distinguishable from profiles of isolates recovered from multiple, unrelated cases of polyarthritis in Queensland and New South Wales and from other countries. All 24 isolates from the clonal cluster possessed a plasmid (pBG7AU) with a molecular size of 1022 bp. DNA sequence analysis of pBG7AU identified two open reading frames sharing 81 and 99% DNA sequence similarity with hypothetical replication control proteins A and B respectively, previously described in plasmid pADB201 isolated from M. mycoides subspecies mycoides. Other isolates of bovine group 7, epidemiologically unrelated to the clonal cluster, including two clones of the type strain PG5O, possessed a similar-sized plasmid. These data confirm that Mycoplasma sp. bovine group 7 is capable of migrating to, and multiplying within, different tissue sites within a single animal and among different animals within a herd.

Genetic diversity amongMycoplasma species bovine group 7: Clonal isolates from an outbreak of polyarthritis, mastitis, and abortion in dairy cattle

A comprehensive genetic analysis of 60 Mycoplasma sp. bovine group 7 isolates from different geographic origins and epidemiological settings is presented. Twenty-four isolates were recovered from the joints of calves during sporadic episodes of polyarthritis in geographically distinct regions of Queensland and New South Wales, Australia, including two clones of the type strain PG5O. A further three Australian isolates were also recovered from the tympanic bulla, retropharyngeal lymph node and the lung and another three isolates had unconfirmed histories. Six isolates originated from Germany, Portugal, Nigeria, and France. Twenty-four epidemiologically related isolates of Mycoplasma sp. bovine group 7 were recovered from multiple tissue sites and body fluids of infected calves with polyarthritis, mastitic milk, and from the stomach contents, lung and liver from aborted foetuses in three large, centrally managed dairy herds in New South Wales, Australia. Restriction endonuclease analysis (REA) of genomic DNA differentiated 29 Cfol profiles among these 60 isolates and grouped all 24 epidemiologically related isolates in a defined pattern showing a clonal origin. Three isolates of this clonal cluster were recovered from mastitic milk and the synovial exudate of clinically-affected calves and appeared sporadically for periods up to 18 months after the initial outbreak of polyarthritis indicating a persistent, close association of the organism with cattle in these herds. The Cfol profile representative of the clonal cluster was distinguishable from profiles of isolates recovered from multiple, unrelated cases of polyarthritis in Queensland and New South Wales and from other countries. All 24 isolates from the clonal cluster possessed a plasmid (pBG7AU) with a molecular size of 1022 bp. DNA sequence analysis of pBG7AU identified two open reading frames sharing 81 and 99% DNA sequence similarity with hypothetical replication control proteins A and B respectively, previously described in plasmid pADB201 isolated from M. mycoides subspecies mycoides. Other isolates of bovine group 7, epidemiologically unrelated to the clonal cluster, including two clones of the type strain PG5O, possessed a similar-sized plasmid. These data confirm that Mycoplasma sp. bovine group 7 is capable of migrating to, and multiplying within, different tissue sites within a single animal and among different animals within a herd.