Proteome alterations during clonal isolation of established human pancreatic cancer cell lines.

Clonal isolation is an integral step of numerous workflows in genome editing and cell engineering. It comprises the isolation of a single progenitor cell from a defined cell line population with subsequent expansion to obtain a monoclonal cell population. This process is associated with transient loss of cell-cell contacts and absence of a multicellular microenvironment. Previous studies have revealed transcriptomic changes upon clonal isolation with cell line specific extent. Since transcriptome alterations are only partially reflected on the proteome level, we sought to investigate the impact of clonal isolation on the cellular proteome to a depth of > 6000 proteins in three established pancreatic cancer cell lines. We show that clonal isolation does have an impact on the cellular proteome, however, with cell line specific extent, affecting different biological processes, and also depending on the isolation method. We demonstrate a different impact of clonal isolation on mesenchymal- and epithelial-derived cell lines mainly affecting cell proliferation, metabolism, cell adhesion and cellular stress. The results bear relevance to the field of genomic editing and cell engineering and highlight the need to consider the impact of clonal isolation when interpreting data stemming from experiments that include this step.

Can Brief Email Guidance Enhance the Effects of an Internet Intervention for People with Problematic Alcohol Use? A Randomized Controlled Trial.

BACKGROUND: Some research suggests that internet interventions aimed at people with problematic alcohol use are more effective when provided with guidance from a therapist or coach. Purpose/Objectives: This trial intended to compare the effects of a previously evaluated internet intervention for people with problematic alcohol use when delivered with or without brief email guidance. Methods: Using online advertising, 238 participants, 18 years or older, were recruited and randomized to receive access to the Internet intervention Alcohol Help Center with or without brief email guidance from a health educator. The guidance consisted of at least four structured, slightly individualized emails delivered during the first two weeks after randomization. Participants were followed up at 3 and 6 months. Results: Number of log-ins did not differ significantly between groups throughout the follow-up period. The follow-up rate at 6 months was 47.0%. Generalized estimating equations run on the primary (standard drinks in preceding week/heavy drinking days in preceding week) and secondary outcome variables (AUDIT, AUDIT-C, quality of life) revealed no significant differences between the interventions on any of the outcomes. Conclusions/Importance: The study does not provide support for any added benefits of providing brief guidance via email in an internet intervention for problem drinkers.

Glomerular development--shaping the multi-cellular filtration unit.

The glomerulus represents a highly structured filtration unit, composed of glomerular endothelial cells, mesangial cells, podocytes and parietal epithelial cells. During glomerulogenesis an intricate network of signaling pathways involving transcription factors, secreted factors and cell-cell communication is required to guarantee accurate evolvement of a functional, complex 3-dimensional glomerular architecture. Here, we want to provide an overview on the critical steps and relevant signaling cascades of glomerular development.

Pathways for development of a biorenewables industry.

The advanced energy initiative to reduce the nation's future demand for oil has resulted in the definition of a number of pathways for the development of the bio-renewables industry. This paper gives an overview of the pathways which could lead to both ethanol and other types of bio-products. The methods that would be used for cellulose conversion also apply to adding value for the co-products of ethanol production. Process milestones and pathways for research that would enable corn dry mill operations to improve are described. A corn dry mill improvement pathway is outlined, and introduces the topics that are covered in this particular special volume.

FERMT2 links cortical actin structures, plasma membrane tension and focal adhesion function to stabilize podocyte morphology.

Simplification and retraction of podocyte protrusions, generally termed as foot process effacement, is a uniform pathological pattern observed in the majority of glomerular disease, including focal segmental glomerulosclerosis. However, it is still incompletely understood how the interaction of cortical actin structures, actomyosin contractility and focal adhesions, is being orchestrated to control foot process morphology in health and disease. By uncovering the functional role of fermitin family member 2 (FERMT2 or kindlin-2) in podocytes, we provide now evidence, how cell-extracellular matrix (ECM) interactions modulate membrane tension and actomyosin contractility. A genetic modeling approach was applied by deleting FERMT2 in a set of in vivo systems as well as in CRISPR/Cas9 modified human podocytes. Loss of FERMT2 results in altered cortical actin composition, cell cortex destabilization associated with plasma membrane blebbing and a remodeling of focal adhesions. We further show that FERMT2 knockout podocytes have high levels of RhoA activation and concomitantly increased actomyosin contractility. Inhibition of actomyosin tension reverses the membrane blebbing phenotype. Thus, our findings establish a direct link between cell-matrix adhesions, cortical actin structures and plasma membrane tension allowing to better explain cell morphological changes in foot process effacement.

The WD40-domain containing protein CORO2B is specifically enriched in glomerular podocytes and regulates the ventral actin cytoskeleton.

Podocytes are highly specialized epithelial cells essentially required to establish and maintain the kidney filtration barrier. Due to their complex cellular architecture these cells rely on an elaborated cytoskeletal apparatus providing plasticity as well as adaptive adhesion properties to withstand significant physical filtration forces. However, our knowledge about podocyte specific components of the cytoskeletal machinery is still incomplete. Employing cross-analysis of various quantitative omics-data sets we identify the WD40-domain containing protein CORO2B as a podocyte enriched protein. Furthermore, we demonstrate the distinct localization pattern of CORO2B to the ventral actin cytoskeleton serving as a physical linkage module to cell-matrix adhesion sites. Analysis of a novel Coro2b knockout mouse revealed that CORO2B modulates stress response of podocytes in an experimental nephropathy model. Using quantitative focal adhesome proteomics we identify the recruitment of CFL1 via CORO2B to focal adhesions as an underlying mechanism. Thus, we describe CORO2B as a novel podocyte enriched protein influencing cytoskeletal plasticity and stress adaptation.

Phenylguanine found in urine after benzene exposure.

Comparative investigations with synthetic N7-phenylguanine were carried out to clarify whether this compound is eliminated via the urine of rats as a benzene-derived nucleic acid adduct. As sensitive methods for detecting trace amounts of the compound, gas chromatography-mass spectroscopy, high performance liquid chromatography, and two immunoassays (enzyme-linked immunosorbent assay and fluoroimmunoassay) with appropriate monoclonal antibodies were used. The results indicate the excretion of several benzene-related guanine adducts slightly different from N7-phenylguanine that may possibly be hydroxylated. These adducts differ also from (O6-, N2- and C8-phenylguanine, respectively.

Production and characterization of monoclonal antibodies to N7-phenylguanine.

N7-Phenylguanine, a base adduct possibly formed after arylation of DNA by benzene oxide, the first reaction metabolite during benzene metabolism, was synthesized in our laboratory and used as reference for the production and characterization of monoclonal antibodies. 2-Hydroxymethyl-7-phenylhypoxanthine, a molecule structurally similar to N7-phenylguanine, was coupled by a linker molecule to different carrier proteins. The resulting conjugate was used to immunize BALB/c mice, the spleen cells of which were fused with mouse P3X63-Ag8.653 myeloma cells to obtain monoclonal antibodies. Several hybridoma lines were cultivated in defined media and characterized as to sensitivity and specificity by an enzyme-linked immunosorbent assay (ELISA). Competitive ELISA demonstrated that all antibodies showed a very high affinity for N7-phenylguanine but had a lower affinity towards various other samples including N7-chlorophenylguanines and C8-, N2-and O6-phenylguanine. As little as about 20 pg N7-phenylguanine could be detected with one of the most sensitive antibodies, CE6/G11, with a colorimetric end point while the detection limit could be lowered to about 10 pg N7-phenylguanine when a fluorescent end point was used. The detection limit of other methods used to determine N7-phenylguanine so far is 10 ng for gas-chromatography/mass-spectrometry and 1 ng for high-pressure liquid chromatography. Thus the use of specific monoclonal antibodies seems to be the most sensitive method for the detection of N7-phenylguanine.

Genotoxic and cytotoxic effects of 4-aroyl-1-nitrosohydrazine-carboxamides on O6-alkylguanine-DNA alkyltransferase-positive and -negative human cell lines.

Five different representatives (I-V) of a new class of bifunctional alkylating agents, the 4-aroyl-1-nitrosohydrazinecarboxamides ("nitrososemicarbazides"), were evaluated for their potential interaction with DNA and for their cytotoxic activity in vitro to O6-alkylguanine-DNA alkyltransferase-positive (Mer+) and -negative (Mer-) human cell lines. The HeLa MR cell line (Mer-) showed up to 20-fold higher sensitivity at IC50 (dose that inhibits colony formation by 50%) to agents I-V than did the HeLa S3 cell line (Mer+) in a colony-formation assay. These data were compared to those obtained by treatment of the two cell lines with carmustine, a currently used antitumor drug. In Mer+ cells comparable results to those with carmustine were obtained with compounds III, IV and V; in Mer- cells compounds I and II showed nearly the same effects as carmustine. Whether compounds I-V produce DNA strand breaks and/or DNA-protein cross-links was investigated using an alkaline filter elution technique. In this assay all compounds produced DNA single-strand breaks; no correlation could be detected between the strand breakage frequency and cytostatic, mutagenic and antitumor activity.

Time course of the effects of steroid hormone deprivation elicited by ovariectomy or ovariectomy plus adrenalectomy on the affinity and density of GABA binding sites in distinct rat brain areas.

The time course of the behaviour of GABAA receptors (affinity of muscimol for GABA binding sites and their number) in membranes obtained from five distinct rat brain areas was evaluated in dependence of the decline of the serum concentrations of estradiol and progesterone following hormone deprivation by either ovariectomy (OVX) or ovary- plus adrenalectomy (OVX-ADX) after 1, 2, 3, 5, 14 and 30 days. Diestrus rats served as control. The effect of OVX on the GABA binding sites was due to a decrease of affinity rather than alterations in their density. Conversely, the combined surgery of OVX plus ADX affected both the affinity and the density. Also, the OVX-linked changes of muscimol binding were observed only after five days as compared to control animals, while the initial OVX-ADX associated changes occurred already after one day. OVX-ADX produced significant decreases in the number of binding sites at the shortest times investigated, in frontal cortex (FC) at day one and two and in the medulla oblongata (MED) after three days, which disappeared in the subsequent times studied. Concomitant with the major decrease of steroid hormone serum levels one day after OVX-ADX the FC displayed an increase in affinity by 45%. From the time when steroid serum levels are low the alterations in affinity elicited by both OVX-ADX and OVX only take a similar course, reflected by the following decrease in affinity. In MED an additional adrenalectomy did not significantly modify the OVX linked decrease of affinity, except that the initial effect was already evident two days after OVX-ADX.(ABSTRACT TRUNCATED AT 250 WORDS)

32P-postlabeling analysis of DNA adducts in different populations.

Blood samples were obtained from different populations exposed occupationally or by lifestyle habits to polycyclic aromatic hydrocarbons (PAH). DNA adducts were determined by 32P-postlabeling assay either in white blood cells (WBC) or lymphocytes. The level of DNA adducts ranged from 1.5 per 10(9) nucleotides in one of the control groups up to 7.1 per 10(9) nucleotides in one group of PAH-exposed workers. Comparison of the adduct thin layer chromatography (TLC) profiles revealed individual variation in both pattern and level of DNA adducts. Significant differences of adduct levels were detected between one group of PAH-exposed coke-oven workers and the corresponding control group. Only a weak influence of the smoking habits on the amount of adducts was detectable in occupationally exposed or unexposed individuals.

Batrachomyia strigapes (Diptera) parasitism of Uperoleia laevigata (Anura).

To quantify the occurrence of the dipteran parasite Batrachomyia strigapes, the ground frog assemblage associated with a large ephemeral wetland in Western Sydney was sampled between January 1999 and April 2000. Parasite infection was restricted to Uperoleia laevigata (5.1%, n = 1,492). Parasites were found under the parotoid glands, most frequently on the left side. After correction for frog size, infection was shown to significantly reduce frog weight. Despite reduced individual fitness, because the level of infection was low in the population, it is unlikely to have a major effect on the anuran population under current environmental conditions.

Report on Gardnerella vaginitis.

Gardnerella vaginitis is one of the most common diagnoses in our outpatient clinic. From January 1, 1989 through July 30, 1989 we had 230 positive cultures for G. vaginitis. Eighty-three compliant patients in this group had unabated symptoms with documented positive cultures for Gardnerella after treatment with metronidazole. This led to the use of alternative regimens of ceftriaxone or clindamycin. A survey of antibiotic choice and post-treatment culture obtained 103 entries from 34 active duty women soldiers and 60 women dependents. This paper presents the results of the survey after an introduction beginning with a 1953 article in U.S. Armed Forces Medical Journal.

Computer searching made easy. Program to find medical articles quickly.

GRATEFUL MED is a computer program for searching the medical literature, designed with the novice in mind. It contains an extensive list of medical subject headings, so the user does not have to page through an index of key words before starting the search. Because of its efficiency and ease of operation, the program is a useful tool for immediate patient care. The program itself is inexpensive, and the hardware needed is minimal.

An association between dentate status and hearing acuity.

Hearing improvement following dental treatment has been reported in case studies, yet few objective studies of this phenomenon have been conducted. To determine if a relationship exists between dentate status and hearing acuity, we conducted a retrospective study of elderly subjects enrolled in a health promotion clinical trial. Air-conduction pure tone average thresholds (PTA) administered to left and right ears at 500 Hz, 1000 Hz, 2000 Hz, 3000 Hz, 4000 Hz, and 6000 Hz were used to create six hearing outcome variables, and were compared between groups of subjects who either had 25 or more teeth (n = 182) or were edentulous (n = 43). Mean PTA thresholds were significantly greater (p < 0.05) in edentulous subjects for each hearing outcome variable, and differences between unadjusted group means ranged from 4.9 dB to 8.6 dB. Mean PTA thresholds adjusted for clinical factors known to affect hearing also indicated worse hearing acuity in the edentulous group. These pilot findings suggest an association between dentate status and hearing acuity.

The different phenotypes of polycystic ovary syndrome: no advantages for identifying women with aggravated insulin resistance or impaired lipids.

BACKGROUND: According to the classification of polycystic ovary syndrome (PCOS) published by the Androgen Excess Society (AES), 10 different phenotypes of the condition are possible. The question remains of whether using these phenotypes might enable us to identify women with impaired insulin resistance or an impaired lipid profile among patients with PCOS. MATERIALS AND METHODS: A prospective cohort analysis was performed of 313 women with diagnosed PCOS and 80 control individuals. The screening panel included a physical examination, weight and height measurement, ultrasonography of the ovaries, and hormone, glucose, lipid, and insulin resistance measurements. RESULTS: There were no statistically significant differences in insulin resistance parameters between the different phenotypes. There were no statistically significant differences in body mass index (BMI) in any of the groups, but BMI showed the best correlation with insulin resistance in all women with PCOS and controls. Sex hormone-binding globulin (SHBG) was inversely correlated with insulin resistance in women with PCOS and controls. High-density lipoprotein (HDL) was negatively correlated with insulin resistance, and free testosterone was positively correlated with it, only in women with PCOS. CONCLUSIONS: Using the different phenotypes described in the AES classification shows no advantages for identifying women with aggravated insulin resistance or impaired lipid profile among patients with PCOS.

15-Deoxy-delta 12-14-prostaglandin-J2 induces hypertrophy and loss of contractility in human testicular peritubular cells: implications for human male fertility.

The wall of the seminiferous tubules contains contractile smooth-muscle-like peritubular cells, thought to be important for sperm transport. Impaired spermatogenesis in men typically involves remodeling of this wall, and we now found that smooth muscle cell (SMC) markers, namely myosin heavy chain (MYH11) and smooth muscle actin (SMA) are often lost or diminished in peritubular cells of testes of men with impaired spermatogenesis. This suggests reduced contractility of the peritubular wall, which may contribute to sub- or infertility. In these cases, testicular expression of cyclooxygenase-2 (COX-2) implies formation of prostaglandins (PGs). When screening different PGs for their ability to target human testicular peritubular cells (HTPCs), only a PG metabolite, 15-deoxy-Delta(12-14)-prostaglandin-J2 (15dPGJ2), was effective. In primary cultures of HTPCs, 15dPGJ2 increased cell size in a reversible manner. Importantly, 15dPGJ2 treatment resulted in a loss of typical differentiation markers for SMCs, namely MYH11, calponin, and SMA, whereas fibroblast markers were unchanged. Collagen gel contraction assays revealed that this loss correlates with a reduced ability to contract. Experiments with an antagonist (bisphenol A diglycidyl ether) and agonist (troglitazone) for a cognate 15dPGJ2 receptor (i.e. peroxisome proliferator-activated receptor-gamma) indicated that peroxisome proliferator-activated receptor-gamma is not directly involved. Rather, the mode of action of 15dPGJ2 involves reactive oxygen species. The antioxidant N-acetylcysteine not only blocked ROS formation but also prevented the increase in cell size and the loss of contractility in HTPCs challenged with 15dPGJ2. We conclude that 15dPGJ2, via reactive oxygen species, influences SMC phenotype and contractility of human peritubular cells and possibly is involved in the development of human male sub-/infertility.