Nine months in space: effects on human autonomic cardiovascular regulation.

We studied three Russian cosmonauts to better understand how long-term exposure to microgravity affects autonomic cardiovascular control. We recorded the electrocardiogram, finger photoplethysmographic pressure, and respiratory flow before, during, and after two 9-mo missions to the Russian space station Mir. Measurements were made during four modes of breathing: 1) uncontrolled spontaneous breathing; 2) stepwise breathing at six different frequencies; 3) fixed-frequency breathing; and 4) random-frequency breathing. R wave-to-R wave (R-R) interval standard deviations decreased in all and respiratory frequency R-R interval spectral power decreased in two cosmonauts in space. Two weeks after the cosmonauts returned to Earth, R-R interval spectral power was decreased, and systolic pressure spectral power was increased in all. The transfer function between systolic pressures and R-R intervals was reduced in-flight, was reduced further the day after landing, and had not returned to preflight levels by 14 days after landing. Our results suggest that long-duration spaceflight reduces vagal-cardiac nerve traffic and decreases vagal baroreflex gain and that these changes may persist as long as 2 wk after return to Earth.     

Different quiescence states of three culture cell lines detected by acridine orange staining of cellular RNA

Three cell lines (CHO, L-929, and R1H) were investigated for their growth kinetics and the difference of exponential and quiescent state of monolayers in medium with and without serum (L-929). The noncycling populations of L-929 and R1H in medium with serum contained increased G1-phase percentages but also considerable proportions of SQ and G2Q cells. Although about 90% of the cells excluded trypan blue, the viability tested by colony assay was clearly lower than for exponentially growing cultures. CHO cells showed similar fractions of cells in G1-, S-, and G2-Q compartments but in addition considerable cell loss. The RNA content of these cells was reduced in plateau phase by 7-48% depending on cell type and residence time in the noncycling state. The data suggest that the cells suffered from nutrition depletion and were arrested in all phases of the cycle. In contrast, L-929 cells in medium without serum reduced their RNA content down to one-third that of proliferating cells and still retained the full viability as shown by the same plating efficiency in a colony assay. Since about 90% of the cells had G1 DNA content, these cells resemble true G1Q or G0 cells controlled by growth factors rather than nutritional depletion.     

Metabolomics enables precision medicine: “A White Paper,Community Perspective”

Introduction: Background to metabolomics

Metabolomics is the comprehensive study of the metabolome, the repertoire of biochemicals (or small molecules) present in cells, tissues, and body fluids. The study of metabolism at the global or “-omics” level is a rapidly growing field that has the potential to have a profound impact upon medical practice. At the center of metabolomics, is the concept that a person’s metabolic state provides a close representation of that individual’s overall health status. This metabolic state reflects what has been encoded by the genome, and modified by diet, environmental factors, and the gut microbiome. The metabolic profile provides a quantifiable readout of biochemical state from normal physiology to diverse pathophysiologies in a manner that is often not obvious from gene expression analyses. Today, clinicians capture only a very small part of the information contained in the metabolome, as they routinely measure only a narrow set of blood chemistry analytes to assess health and disease states. Examples include measuring glucose to monitor diabetes, measuring cholesterol and high density lipoprotein/low density lipoprotein ratio to assess cardiovascular health, BUN and creatinine for renal disorders, and measuring a panel of metabolites to diagnose potential inborn errors of metabolism in neonates.

Objectives of White Paper—expected treatment outcomes and metabolomics enabling tool for precision medicine

We anticipate that the narrow range of chemical analyses in current use by the medical community today will be replaced in the future by analyses that reveal a far more comprehensive metabolic signature. This signature is expected to describe global biochemical aberrations that reflect patterns of variance in states of wellness, more accurately describe specific diseases and their progression, and greatly aid in differential diagnosis. Such future metabolic signatures will: (1) provide predictive, prognostic, diagnostic, and surrogate markers of diverse disease states; (2) inform on underlying molecular mechanisms of diseases; (3) allow for sub-classification of diseases, and stratification of patients based on metabolic pathways impacted; (4) reveal biomarkers for drug response phenotypes, providing an effective means to predict variation in a subject’s response to treatment (pharmacometabolomics); (5) define a metabotype for each specific genotype, offering a functional read-out for genetic variants: (6) provide a means to monitor response and recurrence of diseases, such as cancers: (7) describe the molecular landscape in human performance applications and extreme environments. Importantly, sophisticated metabolomic analytical platforms and informatics tools have recently been developed that make it possible to measure thousands of metabolites in blood, other body fluids, and tissues. Such tools also enable more robust analysis of response to treatment. New insights have been gained about mechanisms of diseases, including neuropsychiatric disorders, cardiovascular disease, cancers, diabetes and a range of pathologies. A series of ground breaking studies supported by National Institute of Health (NIH) through the Pharmacometabolomics Research Network and its partnership with the Pharmacogenomics Research Network illustrate how a patient’s metabotype at baseline, prior to treatment, during treatment, and post-treatment, can inform about treatment outcomes and variations in responsiveness to drugs (e.g., statins, antidepressants, antihypertensives and antiplatelet therapies). These studies along with several others also exemplify how metabolomics data can complement and inform genetic data in defining ethnic, sex, and gender basis for variation in responses to treatment, which illustrates how pharmacometabolomics and pharmacogenomics are complementary and powerful tools for precision medicine.

Conclusions: Key scientific concepts and recommendations for precision medicine

Our metabolomics community believes that inclusion of metabolomics data in precision medicine initiatives is timely and will provide an extremely valuable layer of data that compliments and informs other data obtained by these important initiatives. Our Metabolomics Society, through its “Precision Medicine and Pharmacometabolomics Task Group”, with input from our metabolomics community at large, has developed this White Paper where we discuss the value and approaches for including metabolomics data in large precision medicine initiatives. This White Paper offers recommendations for the selection of state of-the-art metabolomics platforms and approaches that offer the widest biochemical coverage, considers critical sample collection and preservation, as well as standardization of measurements, among other important topics. We anticipate that our metabolomics community will have representation in large precision medicine initiatives to provide input with regard to sample acquisition/preservation, selection of optimal omics technologies, and key issues regarding data collection, interpretation, and dissemination. We strongly recommend the collection and biobanking of samples for precision medicine initiatives that will take into consideration needs for large-scale metabolic phenotyping studies.

     

N-glycosylation Profiling of Colorectal Cancer Cell Lines Reveals Association of Fucosylation with Differentiation and Caudal Type Homebox 1 (CDX1)/Villin mRNA Expression

Various cancers such as colorectal cancer (CRC) are associated with alterations in protein glycosylation. CRC cell lines are frequently used to study these (glyco)biological changes and their mechanisms. However, differences between CRC cell lines with regard to their glycosylation have hitherto been largely neglected. Here, we comprehensively characterized the N-glycan profiles of 25 different CRC cell lines, derived from primary tumors and metastatic sites, in order to investigate their potential as glycobiological tumor model systems and to reveal glycans associated with cell line phenotypes. We applied an optimized, high-throughput membrane-based enzymatic glycan release for small sample amounts. Released glycans were derivatized to stabilize and differentiate between α2,3- and α2,6-linked N-acetylneuraminic acids, followed by N-glycosylation analysis by MALDI-TOF(/TOF)-MS. Our results showed pronounced differences between the N-glycosylation patterns of CRC cell lines. CRC cell line profiles differed from tissue-derived N-glycan profiles with regard to their high-mannose N-glycan content but showed a large overlap for complex type N-glycans, supporting their use as a glycobiological cancer model system. Importantly, we could show that the high-mannose N-glycans did not only occur as intracellular precursors but were also present at the cell surface. The obtained CRC cell line N-glycan features were not clearly correlated with mRNA expression levels of glycosyltransferases, demonstrating the usefulness of performing the structural analysis of glycans. Finally, correlation of CRC cell line glycosylation features with cancer cell markers and phenotypes revealed an association between highly fucosylated glycans and CDX1 and/or villin mRNA expression that both correlate with cell differentiation. Together, our findings provide new insights into CRC-associated glycan changes and setting the basis for more in-depth experiments on glycan function and regulation.Colorectal cancer (CRC)¹ is a very prevalent and heterogeneous pathology with highly variable disease progression and clinical outcome among patients. It is the third most common cancer in men and the second most common in women (1) with a highly stage-specific patient survival (2). Treatments are often curative for patients with local disease stages (stage I-II), whereas a 5-year survival of only 13% is observed in patients with distant metastasis (stage IV) (2). As CRC is often asymptomatic in the first years, unfortunately, only 40% of the patients are diagnosed at stage I-II, thus pointing to the urgent need of sensitive diagnostic tools for early detection and consequently effective, curative treatment (3). In this context, understanding the complex mechanisms of CRC is an overriding condition for the development of new, more efficient means of detection, treatment, and prognosis of the disease.Altered glycosylation is a hallmark of cancer (4) and is known to occur with cancer progression (4, 5) as glycans are involved in many cancer-associated events such as adhesion, invasion, and cell signaling (6). As a result of altered glycan structures, cellular processes can be affected due to a change of interactions with glycan-binding proteins (7–9). Several CRC tissue-associated changes in N-glycans, O-glycans, and glycosphingolipid glycans have been reported and recently reviewed (7). For instance, N-glycans extracted from colorectal tumor tissues are characterized by an increase of sulfated glycans, (truncated) high-mannose-type glycans, and glycans containing sialylated Lewis type epitopes, while showing a decrease of bisection as compared with glycans from nontumor colorectal tissue of the same individuals (10). In accordance, elevated expression of sialyl Lewis A (NeuAcα2,3Galβ1,3[Fucα1,4]GlcNAc-R; NeuAc = N-acetylneuraminic acid, Gal = galactose, Fuc = fucose, GlcNAc = N-acetylglucosamine, R = rest) and pauci-mannosidic N-glycans (truncated high-mannose-type, Man1–4GlcNAc1–4GlcNAc; Man = mannose) was recently found to be correlated with poor prognosis in (advanced) colon carcinomas and N-glycomic profiling was successfully applied to distinguish colorectal adenomas from carcinomas (11).Due to limitations in accessibility of tumor materials and possibilities of in vivo studies on a large scale, cancer cell lines represent a relevant alternative and are widely used as model systems for studying the molecular mechanisms associated with cancer outcome and progression. Since the early 1960s, colorectal cancer cell lines have been established with HT29, LoVo, LS-180, LS-174T, and Co115 representing the first continuous cell lines derived from colon tumors and xenografts (12–14). Major benefits of cancer cell lines are their continuous availability, their fast growth, and relatively easy handling, making them suitable also for high-throughput screenings (15) and a large range of experimental possibilities (16). Of note, advantages and limitations of cell lines have been recently reviewed (15).In order to select suitable in vitro models, the characterization of molecular features and their comparison to tumor tissues are needed. A detailed Cancer Cell Line Encyclopedia was recently established containing a genomic dataset for 947 human cancer cell lines, from which 58 are colorectal cancer lineages (17). The Cancer Cell Line Encyclopedia includes data collections on genomic characterization, point mutation frequencies, DNA copy number, and mRNA expression levels. Comparison of these features between cell lines and primary tumors showed a high correlation in most cancer types, especially for colorectal cancer, suggesting that cell lines do represent tumor tissues quite reasonably at least on the genetic level. However, the number of publications characterizing cancer cell lines at a molecular level is far behind the number of articles using cancer cell lines as model systems (18), and only few studies have been conducted on whether in vitro cultured cell lines can serve as suitable models for human tumors (19–22). Furthermore, cell lines are well characterized genetically, but they are largely understudied with regard to their glycosylation profiles.Here, we developed and optimized a new analytical method for the more sensitive and higher throughput N-glycome profiling of cells. This method is based on the release of N-glycans in a 96-well plate format from a PVDF-membrane (23) starting from a low number of cells (250,000 cells), the chemical derivatization of released N-glycans enabling the stabilization and discrimination of α2,3- and α2,6-linked N-acetylneuraminic acids (24), followed by registration of the N-glycans by MALDI-TOF(/TOF)-MS. The method was applied to characterize the N-glycome of 25 different colorectal cell lines in a fast and robust manner, including biological and technical replicates for all the cell lines. We obtained the comprehensive N-glycan profiles of 21 cell lines derived from primary tumors, two from lymph node metastases, one from a lung metastasis, and one from ascites fluid to assess their potential as glycobiological tumor model systems. Cancer cell line glycosylation features were then correlated with cancer cell markers and phenotypes as well as glycosyltransferase expressions. This study provides new insights into colon-cancer-associated glycan changes and sets a basis for studies into the functions of N-glycans in CRC with cell lines as model systems.     

RNA‐directed DNA methylation efficiency depends on trigger and target sequence identity

RNA‐directed DNA methylation (RdDM) in plants has been extensively studied, but the RNA molecules guiding the RdDM machinery to their targets are still to be characterized. It is unclear whether these molecules require full complementarity with their target. In this study, we have generated Nicotiana tabacum (Nt) plants carrying an infectious tomato apical stunt viroid (TASVd) transgene (Nt‐TASVd) and a non‐infectious potato spindle tuber viroid (PSTVd) transgene (Nt‐SB2). The two viroid sequences exhibit 81% sequence identity. Nt‐TASVd and Nt‐SB2 plants were genetically crossed. In the progeny plants (Nt‐SB2/TASVd), deep sequencing of small RNAs (sRNAs) showed that TASVd infection was associated with the accumulation of abundant small interfering RNAs (siRNAs) that mapped along the entire TASVd but only partially matched the SB2 transgene. TASVd siRNAs efficiently targeted SB2 RNA for degradation, but no transitivity was detectable. Bisulfite sequencing in the Nt‐SB2/TASVd plants revealed that the TASVd transgene was targeted for dense cis‐RdDM along its entire sequence. In the same plants, the SB2 transgene was targeted for trans‐RdDM. The SB2 methylation pattern, however, was weak and heterogeneous, pointing to a positive correlation between trigger–target sequence identity and RdDM efficiency. Importantly, trans‐RdDM on SB2 was also detected at sites where no homologous siRNAs were detected. Our data indicate that RdDM efficiency depends on the trigger–target sequence identity, and is not restricted to siRNA occupancy. These findings support recent data suggesting that RNAs with sizes longer than 24 nt (>24‐nt RNAs) trigger RdDM.     

Uptake of HCO3? and CO2 in Cells and Chloroplasts from the Microalgae Chlamydomonas reinhardtii and Dunaliella tertiolecta

Mass-spectrometric disequilibrium analysis was applied to investigate CO₂ uptake and HCO₃⁻ transport in cells and chloroplasts of the microalgae Dunaliella tertiolecta and Chlamydomonas reinhardtii, which were grown in air enriched with 5% (v/v) CO₂ (high-Ci cells) or in ambient air (low-Ci cells). High- and low-Ci cells of both species had the capacity to transport CO₂ and HCO₃⁻, with maximum rates being largely unaffected by the growth conditions. In high- and low-Ci cells of D. tertiolecta, HCO₃⁻ was the dominant inorganic C species taken up, whereas HCO₃⁻ and CO₂ were used at similar rates by C. reinhardtii. The apparent affinities of HCO₃⁻ transport and CO₂ uptake increased 3- to 9-fold in both species upon acclimation to air. Photosynthetically active chloroplasts isolated from both species were able to transport CO₂ and HCO₃⁻. For chloroplasts from C. reinhardtii, the concentrations of HCO₃⁻ and CO₂ required for half-maximal activity declined from 446 to 33 μm and 6.8 to 0.6 μm, respectively, after acclimation of the parent cells to air; the corresponding values for chloroplasts from D. tertiolecta decreased from 203 to 58 μm and 5.8 to 0.5 μm, respectively. These results indicate the presence of inducible high-affinity HCO₃⁻ and CO₂ transporters at the chloroplast envelope membrane.     

GCAC1 recognizes the pH gradient across the plasma membrane: a pH-sensitive and ATP-dependent anion channel links guard cell membrane potential to acid and energy metabolism

Ion channels in the plasma membrane of guard cells provide key mechanisms in signal perception and volume regulation during stomatal movement. Recent studies have suggested that the strongly voltage-dependent, inactivating g uard c ell a nion c hannel (GCAC1) acts as a sensor of the ambient extracellular CO₂ concentration and as a target of modulation by nucleotides and Ca²⁺ ions. Applying the patch-clamp technique it is demonstrated here that GCAC1 is activated by cytoplasmic ATP in a pH-dependent manner. When the apoplastic pH was buffered to 5.6 and the cytosolic pH dropped step-wise from 7.8 to 5.6, the single-channel activity increased as a function of proton concentration. This pH-sensitivity is characterized by a titratable site with an apparent pK value around 6.9. While the steepness and direction of the transmembrane pH gradient did not affect the kinetics of activation, deactivation and fast inactivation of the whole-cell anion current, the kinetics of slow inactivation and reactivation were strongly influenced. When at a constant intracellular proton concentration of pH 7.2 the external pH decreased from 7.2 to 5.6 the time constants of slow inactivation and the half-times of reactivation increased two- and sevenfold, respectively. The mechanism of nucleotide activation of GCAC1 was quantitatively analysed on the level of single-channel events. Using inside-out, cell-free membrane patches, GCAC1 half-activated around 0.4 mM ATP. The sigmoidal dose-dependence of anion channel activation could be well fitted with an apparent Hill coefficient of 3.6. This behaviour might indicate that the activation process involves a strongly cooperative interaction of four ATP-binding sites. Neither ATP nor its non-hydrolysable analogue AMP-PMP, which also activated GCAC1, altered the voltage-dependent gating. AMP-PMP stimulation and the insensitivity of GCAC1 towards the phosphatase inhibitor, okadaic acid, and the kinase inhibitors, staurosporine and H-7, provided evidence that nucleotide binding rather than phosphorylation caused channel activation. Since the time- and voltage-dependent activity of GCAC1 is strongly modulated by ATP and protons, this channel is capable of sensing changes in the energy status, acid metabolism and the H⁺ ATPase activity of guard cells.     

Intratumoral heterogeneity of S phase transition in solid tumours determined by bromodeoxyuridine labelling and flow cytometry

Abstract. Cell kinetics of human renal cell carcinomas xenotransplanted into nu/nu mice were analysed using the bromodeoxyuridine (BrdUrd) labelling method. Tumours were removed 0.5–14 h after injection of the BrdUrd solution. The tumour cells were stained with fluorescein isothiocyanate conjugated anti-BrdUrd antibodies and propidium iodide (DNA content). From the flow cytometry data the relative movement was calculated. Relative movement data of variable intervals after BrdUrd labelling were subjected to a fit procedure using log-normal distributions for S phase transition (T_s). The log-normal distributions were modified by inflation factors in order to get extremely asymmetric distributions. The best fits to the experimental data were obtained using wide asymmetric T_s distributions, indicating that progression through S phase in solid human tumours is considerably heterogeneous. This implies that the potential doubling time (T_pot) is longer than calculated from a single measured relative movement value obtained a few hours after BrdUrd labelling.     

Molecular analysis of a gene fromBacteroides fragilis involved in metronidazole resistance inEscherichia coli

The region ofBacteroides fragilis DNA on the recombinant plasmid pMT100 responsible for conferring metronidazole resistance inEscherichia coli strains was characterized. An open reading frame (ORF1) of 195 bp encoded a protein of 64 amino acids with a predicted M_r, of 7.3 kDa. Deletion analysis indicated that ORF1 conferred the metronidazole resistance phenotype and encoded a protein with an apparent M_r of approximately 8–10 kDa.