Methods to capture proteomic and metabolomic signatures from cerebrospinal fluid and serum of healthy individuals.

Discovery of reliable signatures for the empirical diagnosis of neurological diseases-both infectious and non-infectious-remains unrealized. One of the primary challenges encountered in such studies is the lack of a comprehensive database representative of a signature background that exists in healthy individuals, and against which an aberrant event can be assessed. For neurological insults and injuries, it is important to understand the normal profile in the neuronal (cerebrospinal fluid) and systemic fluids (e.g., blood). Here, we present the first comparative multi-omic human database of signatures derived from a population of 30 individuals (15 males, 15 females, 23-74 years) of serum and cerebrospinal fluid. In addition to empirical signatures, we also assigned common pathways between serum and CSF. Together, our findings provide a cohort against which aberrant signature profiles in individuals with neurological injuries/disease can be assessed-providing a pathway for comprehensive diagnostics and therapeutics discovery.

Progress Toward a Multiomic Understanding of Traumatic Brain Injury: A Review.

Traumatic brain injury (TBI) is not a single disease state but describes an array of conditions associated with insult or injury to the brain. While some individuals with TBI recover within a few days or months, others present with persistent symptoms that can cause disability, neuropsychological trauma, and even death. Understanding, diagnosing, and treating TBI is extremely complex for many reasons, including the variable biomechanics of head impact, differences in severity and location of injury, and individual patient characteristics. Because of these confounding factors, the development of reliable diagnostics and targeted treatments for brain injury remains elusive. We argue that the development of effective diagnostic and therapeutic strategies for TBI requires a deep understanding of human neurophysiology at the molecular level and that the framework of multiomics may provide some effective solutions for the diagnosis and treatment of this challenging condition. To this end, we present here a comprehensive review of TBI biomarker candidates from across the multiomic disciplines and compare them with known signatures associated with other neuropsychological conditions, including Alzheimer's disease and Parkinson's disease. We believe that this integrated view will facilitate a deeper understanding of the pathophysiology of TBI and its potential links to other neurological diseases.

Breakdown of multiple sclerosis genetics to identify an integrated disease network and potential variant mechanisms.

Genetics of multiple sclerosis (MS) are highly polygenic with few insights into mechanistic associations with pathology. In this study, we assessed MS genetics through linkage disequilibrium and missense variant interpretation to yield a MS gene network. This network of 96 genes was taken through pathway analysis, tissue expression profiles, single cell expression segregation, expression quantitative trait loci (eQTLs), genome annotations, transcription factor (TF) binding profiles, structural genome looping, and overlap with additional associated genetic traits. This work revealed immune system dysfunction, nerve cell myelination, energetic control, transcriptional regulation, and variants that overlap multiple autoimmune disorders. Tissue-specific expression and eQTLs of MS genes implicate multiple immune cell types including macrophages, neutrophils, and T cells, while the genes in neural cell types enrich for oligodendrocyte and myelin sheath biology. There are eQTLs in linkage with lead MS variants in 25 genes including the multitissue eQTL, rs9271640, for HLA-DRB1/DRB5. Using multiple functional genomic databases, we identified noncoding variants that disrupt TF binding for GABPA, CTCF, EGR1, YY1, SPI1, CLOCK, ARNTL, BACH1, and GFI1. Overall, this paper suggests multiple genetic mechanisms for MS associated variants while highlighting the importance of a systems biology and network approach when elucidating intersections of the immune and nervous system.

A candidate gene approach identifies variants in SLC45A2 that explain dilute phenotypes, pearl and sunshine, in compound heterozygote horses.

Variations in the SLC45A2 gene are responsible for the dilution phenotypes cream and pearl in domestic horses. Cream dilution is inherited in an incomplete dominant manner, diluting only red in the heterozygous state but both red and black pigments when two alleles are present. The pearl dilution is recessive and dilutes only the red and black pigment in the homozygous state or when paired with a cream allele. Horses that inherit one copy of pearl (Cprl ) and one copy of the dominant cream allele (CC r ) display a dilution phenotype similar to that of homozygous cream, suggesting that pearl is the result of a different variation in the same gene responsible for cream. We sequenced SLC45A2 in two 'false double dilute' horses that appeared phenotypically homozygous cream but tested as possessing only a single CC r allele. We also sequenced one known pearl carrier to screen for putative causal variants. The missense variant ECA21:SLC45A2:c.985G>A; p.Ala329Thr (Cprl ) was present in one false double dilute and the pearl carrier and was also genotyped in an additional 126 horses for statistical evaluation. The genotype matched the expected phenotype in all horses (P-value = 6.5 × 10-41 ) and is identical to a pearl variant found previously. The second false double dilute horse and one non-dilute offspring genotyped as heterozygous for a novel missense variant ECA21:SLC45A2:c.568G>A (p.Gly190Arg), the proposed Csun variant (for the name of the horse). This variant produces a recessive dilution similar to pearl and indicates that multiple alleles of SLC45A2 result in dilution phenotypes in the domestic horse.

Correction to: The SAMHD1-mediated block of LINE-1 retroelements is regulated by phosphorylation.

[This corrects the article DOI: 10.1186/s13100-018-0116-5.].

Limited nucleotide pools restrict Epstein-Barr virus-mediated B-cell immortalization.

Activation of cellular oncogenes as well as infection with tumor viruses can promote aberrant proliferation and activation of the host DNA damage response. Epstein-Barr virus (EBV) infection of primary human B cells induces a transient period of hyper-proliferation, but many of these infected cells succumb to an ataxia telangiectasia mutated/checkpoint kinase 2 (ATM/Chk2)-mediated senescence-like growth arrest. In this study, we assessed the role of DNA replicative stress and nucleotide pool levels in limiting EBV-infected B-cell outgrowth. We found that EBV triggered activation of the ataxia telangiectasia and Rad3-related (ATR) signaling pathway in the early rapidly proliferating cells, which were also significantly more sensitive to inhibition of the ATR pathway than late attenuated proliferating cells. Through nuclear halo assays, we determined that early EBV-infected cells displayed increased replicative stress and DNA damage relative to late proliferating cells. Finally, we found that early after infection, hyper-proliferating B cells exhibited limited deoxyribonucleotide triphosphate (dNTP) pools compared with late proliferating and EBV-immortalized lymphoblastoid cell lines with a specific loss of purine dNTPs. Importantly, supplementation with exogenous nucleosides before the period of hyper-proliferation markedly enhanced B-cell immortalization by EBV and rescued replicative stress. Together our results suggest that purine dNTP biosynthesis has a critical role in the early stages of EBV-mediated B-cell immortalization.

Co-culturing human prostate carcinoma cells with hepatocytes leads to increased expression of E-cadherin.

Metastasis is a multi-step process wherein tumour cells detach from the primary mass, migrate through barrier matrices, gain access to conduits to disseminate, and subsequently survive and proliferate in an ectopic site. During the initial invasion stage, prostate carcinoma cells undergo epithelial-mesenchymal-like transition with gain of autocrine signalling and loss of E-cadherin, hallmarks that appear to enable invasion and dissemination. However, some metastases express E-cadherin, and we found close connections between prostate carcinoma cells and hepatocytes in a liver microtissue bioreactor. We hypothesise that phenotypic plasticity occurs late in prostate cancer progression at the site of ectopic seeding. Immunofluorescence staining for E-cadherin in co-cultures of hepatocytes and DU-145 prostate cancer cells revealed E-cadherin upregulation at peripheral sites of contact by day 2 of co-culture; E-cadherin expression also increased in PC-3 cells in co-culture. These carcinoma cells bound to hepatocytes in an E-cadherin-dependent manner. Although the signals by which the hepatocytes elicited E-cadherin expression remain undetermined, it appeared related to downregulation of epidermal growth factor receptor (EGFR) signalling. Inhibition of autocrine EGFR signalling increased E-cadherin expression and cell-cell heterotypic adhesion; further, expression of a downregulation-resistant EGFR variant prevented E-cadherin upregulation. These findings were supported by finding E-cadherin and catenins but not activated EGFR in human prostate metastases to the liver. We conclude that the term epithelial-mesenchymal transition only summarises the transient downregulation of E-cadherin for invasion with re-expression of E-cadherin being a physiological consequence of metastatic seeding.

PLC gamma contributes to metastasis of in situ-occurring mammary and prostate tumors.

Phospholipase C-gamma (PLCgamma) has been implicated in tumor cell motility required for invasiveness and metastasis. Diminished tumor dissemination has been demonstrated in xenograft models, but studies in naturally-occurring tumors are lacking, having been limited by the timing of the interventions. Therefore, we generated mice that express a doxycycline (DOX)-inducible dominant-negative fragment of PLCgamma, PLCz; this approach avoids the in utero lethality caused by the absence of PLCgamma. As we targeted two de novo-occurring carcinomas of the mammary (MMTV-driven polyoma middle T antigen model, PyVmT) and prostate (TRAMP model) glands, we limited expression to these epithelial cells by driving DOX transactivator from the prostatein C3 promoter. This avoids the confounding variable of potentially abrogating motility in stromal and endothelial cells. These mice developed normally in the presence of DOX, except for limited mammary development if treated before 6 weeks and immaturity of the prostate gland if treated before 2 weeks of age. DOX-mediated induction of PLCz from age 8 to 16 weeks in PyVmT mice decreased the number of lung metastases by >10-fold (P<0.06) without a detectable effect on in situ tumor cell proliferation or tumor size. Lung metastases were also significantly decreased in the TRAMP model in which the mice expressed the PLCz fragment (P<0.05). DOX treatment itself had no effect on tumor size or metastasis in control mice, nor did it affect tumor dissemination in nontransgenic littermates. In conclusion, abrogation of the PLCgamma signaling pathway can limit the metastatic potential of carcinomas.

Bioterrorism-related inhalational anthrax: the first 10 cases reported in the United States.

From October 4 to November 2, 2001, the first 10 confirmed cases of inhalational anthrax caused by intentional release of Bacillus anthracis were identified in the United States. Epidemiologic investigation indicated that the outbreak, in the District of Columbia, Florida, New Jersey, and New York, resulted from intentional delivery of B. anthracis spores through mailed letters or packages. We describe the clinical presentation and course of these cases of bioterrorism-related inhalational anthrax. The median age of patients was 56 years (range 43 to 73 years), 70% were male, and except for one, all were known or believed to have processed, handled, or received letters containing B. anthracis spores. The median incubation period from the time of exposure to onset of symptoms, when known (n=6), was 4 days (range 4 to 6 days). Symptoms at initial presentation included fever or chills (n=10), sweats (n=7), fatigue or malaise (n=10), minimal or nonproductive cough (n=9), dyspnea (n=8), and nausea or vomiting (n=9). The median white blood cell count was 9.8 X 10(3)/mm(3) (range 7.5 to 13.3), often with increased neutrophils and band forms. Nine patients had elevated serum transaminase levels, and six were hypoxic. All 10 patients had abnormal chest X-rays; abnormalities included infiltrates (n=7), pleural effusion (n=8), and mediastinal widening (seven patients). Computed tomography of the chest was performed on eight patients, and mediastinal lymphadenopathy was present in seven. With multidrug antibiotic regimens and supportive care, survival of patients (60%) was markedly higher (<15%) than previously reported.

Stress measurements in glass by use of double thermal gratings.

We developed a nondestructive and noncontact method for measuring stress at the midplane of tempered glass plates that uses Bragg scattering from a pair of thermal gratings. These gratings are formed by 1064-nm beams from a seeded Nd:YAG laser, and we measure the polarization state of light from a 532-nm beam that scatters from both thermal gratings. The change in polarization of the doubly scattered light with separation between the two gratings allows measurement of the in-plane stress. A model of the Bragg scattering efficiency, experimental investigations of the scattered beams, and stress measurements are reported.

Fatigue and other symptoms in patients with chronic obstructive pulmonary disease: do women and men differ?

OBJECTIVE: Describe the symptoms experienced by patients with chronic obstructive pulmonary disease (COPD), compare women and men with regard to their physical and psychologic symptoms, and determine the predictors of fatigue for the group as a whole and for women separately. DESIGN: Correlational, predictive. PARTICIPANTS: One hundred four (48 women/56 men) outpatients with severe COPD and a mean age of 60 years. MEASURES: Self-report of fatigue using the SF-36 energy/fatigue subscale and a "lack of energy" question. Self-report of dyspnea, general symptoms using the Memorial Symptom Assessment Scale, and psychologic symptoms using the Brief Symptom Inventory. Pulmonary function tests and arterial blood gas levels. RESULTS: Patients reported a moderate level of fatigue (mean of 44 on the SF-36), with no difference in the reports of men and women. When reporting their "lack of energy" during the past week, women reported more fatigue than did men. Other symptoms in descending order of reporting were dyspnea and cough. More women reported "don't look like myself" than did men. Women and men were similar in their psychologic symptoms except anxiety, which was higher in women (t = 2.64, p < .01). Predictors of fatigue (SF-36 subscale) for the group as a whole were found to be dyspnea and physical symptoms, predicting 42% of the variance. For the women alone, dyspnea and physical symptoms entered the equation, to predict 67% of the variance in fatigue. CONCLUSIONS: Predictors of fatigue were similar for women compared to the group as a whole, with symptoms having more of an impact on fatigue than did physiologic variables.

Measurements of density and viscosity of one- and two-phase fluids with torsional waveguides.

The continuing need for in-situ measurements of physical properties of wastes contained within many high level radioactive waste tanks within the Hanford Site has initiated experimental and theoretical investigations of candidate measurement methods. This paper describes experiments performed with acoustic waveguide sensors. This technology has potential application at the Hanford Site for in-situ measurements of density, viscosity, and temperature of liquid wastes. Waveguides of both circular and rectangular geometry were used in these studies for determination of the densities and viscosities of various fluids. The flight time of a torsional pulse through the sensing region of the waveguide forms the measured quantity. The flight time depends on the velocity of the wave through the sensing region of the waveguide, and this velocity in turn depends upon the properties of the fluid in contact with the waveguide. We performed experiments with 15 different fluids, most of which were single-phase Newtonian fluids. However, three of the fluids were particle-liquid mixtures, and one of these Newtonian in behavior. Most of the wastes held in Hanford tanks contain high solids content. The results of our experiments showed that acoustic waveguides were well suited for measurements in most Newtonian fluids, in agreement with earlier research presented in the literature. However, results for two-phase Newtonian fluids containing particles indicate that, in our case, the waveguides responded primarily to the background fluid rather than the mixture. Very poor results were obtained with the non-Newtonian fluid. In addition, there was a class of fluids, which serve the community as viscosity standards, for which viscosities determined with torsional waveguides were in disagreement with viscosities obtained with standard viscometers.

HIV serology in artificially inseminated lesbians.

The purpose of this investigation was to examine the risks of HIV transmission from artificial insemination in a sample of lesbians residing in California and inseminated between 1979 and 1987. This population was selected because lesbians are considered to be at low risk for HIV infection, and have traditionally used semen from men engaging in high-risk behaviors, namely, homosexual men. Each of the 98 participants had blood drawn for the HIV antibody test (ELISA) and completed a questionnaire inquiring about her sexual, health, and reproductive history, including detailed information about her inseminations (e.g., vaginal vs. uterine, fresh vs. frozen semen, sexual orientation, and antibody status of donor). One-half of the women had homosexual or bisexual donors, many of whom resided in the San Francisco Bay area; most of these inseminations were with fresh semen. The women reported a marked decline in the use of homosexual donors after 1982 and a corresponding increase in the use of donations from sperm banks and health practitioners. Based on the women's reports, as many as 11 women may have received semen from an infected donor. However, none of the 98 women tested seropositive. We attribute our negative findings to the change to low-risk donors in the years when HIV became more prevalent in the population, and to the potentially lower rates of infectivity with artificial insemination than with heterosexual intercourse. Nevertheless, we recommend that women continue to follow the CDC guidelines for screening donors prior to artificial insemination.

Computerized interactive morphometry of brushing cytology specimens.

Forty-two bronchial brushing cytology specimens were evaluated by a video-based computerized interactive morphometry (CIM) system with an interactive peripheral consisting of a touch-sensitive screen mounted over a high-resolution video monitor. The system was programmed to allow a trained observer to rapidly measure nuclear and cytoplasmic profile diameters of randomly selected cells and to calculate their nuclear-cytoplasmic ratios. The specimens included 13 cytologic slides with no malignant cells present, 14 with non-small cell carcinoma cells and 15 with small cell carcinoma cells. The cases were divided into two groups: a training set composed of slides with "known diagnosis" and a test set of slides with "unknown diagnosis". A data set was constructed with the measurements from the cases with "known diagnosis," and an algorithm that allowed the classification of cases by hierarchical analysis was developed. The data was also analyzed with statistical methods of classificatory discriminant analysis. Utilizing the information in the data base, the slides with "unknown diagnosis" were classified individually; all cases were correctly classified by the procedures. Potential applications of CIM in cytology are discussed.

Activity of selected beta-lactam antibiotics against Mycobacterium leprae.

Twelve beta-lactam antibiotics were tested for activity against Mycobacterium leprae growing in the foot pads of mice. Two cephalosporins (7-aminocephalosporanic acid and cefuroxime) and one cephamycin (cefoxitin) showed significant activity against M. leprae, and one penicillin (mezlocillin) exerted possible growth-promoting activity. These results suggest that particular molecular structures may be required for activity against M. leprae.

Susceptibility of strains of Mycobacterium leprae isolated prior to 1977 from patients with previously untreated lepromatous leprosy.

Because of the recent spate of reports of primary resistance to dapsone among patients with lepromatous leprosy, largely to small concentrations of the drug, a survey was made of the results of dapsone-susceptibility testing of strains of Mycobacterium leprae isolated before 1977 among six laboratories which employed the mouse foot pad technique for this work prior to that time. Data have been found for strains that had been isolated from 73 patients, representing 19 countries and dependencies, with previously untreated lepromatous leprosy; all 73 strains were inhibited from multiplication by dapsone administered to mice in a concentration of 0.0001 g per 100 g mouse diet. These data suggest that the properties of M. leprae isolated from previously untreated patients with respect to susceptibility to dapsone have changed since the years preceding 1977.