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The intact polar lipid (IPL) composition of phytoplankton is plastic and dependent on environmental factors. Previous studies have shown that phytoplankton under phosphorus (P)-stress substitute phosphatidylglycerols (PGs) with sulphoquinovosyldiacylglycerols (SQDGs) and digalactosyldiacylglycerols (DGDGs). However, these studies focused merely on P-depletion, while phytoplankton in the natural environment often experience P-limitation whereby the degree of limitation depends on the supply rate of the limiting nutrient. Here we demonstrate a linear increase in SQDG : PG and DGDG : PG ratios with increasing cellular P-stress in the picophotoeukaryote Micromonas pusilla, obtained by P-replete, P-limited (chemostat) and P-starved (no supply of P) culturing conditions. These ratios were not affected by the degree of the P-limiting conditions itself (i.e. 0.97 and 0.32 µmax chemostats), suggesting there is a minimum requirement of PGs for the maintenance of cell growth. Viral infection reduced the increase in SQDG : PG and DGDG : PG ratios in P-starved cells, but the extent did depend on the growth rate of the cultures before infection. The membrane of M. pusilla virus MpV itself was lacking some IPLs compared to the host as, e.g. no monogalactosyldiacylglycerols could be detected. Growth of the phytoplankton cultures under enhanced CO2 concentration did not affect the lipid remodeling results. The present study provides new insights into how the P-related trophic state of an ecosystem as well as viral infection can affect phytoplankton IPL composition, and therefore influence food web dynamics and biogeochemical cycling. [ABSTRACT FROM AUTHOR]

Recent studies showed changes in phytoplankton lipid composition during viral infection and have indicated roles for specific lipids in the mechanisms of algal virus-host interaction. To investigate the generality of these findings and obtain a better understanding of the allocation of specific lipids to viruses, we studied the intact polar lipid (IPL) composition of virally infected and non-infected cultures of the prymnesiophyte Phaeocystis globosa G(A) and its lytic virus PgV-07T. The P. globosa IPL composition was relatively stable over a diel cycle and not strongly affected by viral infection. Glycolipids, phospholipids and betaine lipids were present in both the host and virus, although specific groups such as the diacylglyceryl-hydroxymethyltrimethyl-ß-alanines and the sulfoquinovo-syldiacylglycerols, were present in a lower proportion or were not detected in the virus. Viral glycosphingolipids (vGSLs), which have been shown to play a role in the infection strategy of the virus EhV-86, infecting the prymnesiophyte Emiliania huxleyi CCMP374, were not encountered. Our results show that the involvement of lipids in virus-algal host interactions can be very different amongst virus-algal host systems. [ABSTRACT FROM AUTHOR]

Recent studies showed changes in phytoplankton lipid composition during viral infection and have indicated roles for specific lipids in the mechanisms of algal virus-host interaction. To investigate the generality of these findings and obtain a better understanding of the allocation of specific lipids to viruses, we studied the intact polar lipid (IPL) composition of virally infected and non-infected cultures of the Prymnesiophyte Phaeocystis globosa G(A) and its lytic virus PgV-07T. The P globosa IPL composition was relatively stable over a diel cycle and not strongly affected by viral infection. Glycolipids, phospholipids and betaine lipids were present in both the host and virus, although specific groups such as the diacylglyceryl-hydroxymethyltrimethyl-β-alanines and the sulfoquinovosyldiacylglycerols, were present in a lower proportion or were not detected in the virus. Viral glycosphingolipids (vGSLs), which have been shown to play a role in the infection strategy of the virus EhV-86, infecting the Prymnesiophyte Emiliania huxleyi CCMP374, were not encountered. Our results show that the involvement of lipids in virus-algal host interactions can be very different amongst virus-algal host systems. [ABSTRACT FROM AUTHOR]

The intact polar lipid ( IPL) composition of twelve hydrothermal vent deposits from the Rainbow ( RHF) and Lucky Strike hydrothermal fields ( LSHF) has been investigated in order to assess its utility as a proxy for microbial community composition associated with deep-sea hydrothermal locations. Gene-based culture-independent surveys of the microbial populations of the same vent deposits have shown that microbial populations are different in the two locations and appear to be controlled by the geochemical and geological processes that drive hydrothermal circulation. Large differences in the IPL composition between these two sites are evident. In the ultramafic-hosted RHF, mainly archaeal- IPLs were identified, including those known to be produced by hyperthermophilic Euryarchaeota. More specifically, polyglycosyl derivatives of archaeol and macrocyclic archaeol indicate the presence of hyperthermophilic methanogenic archaea in the vent deposits, which are related to members of the Methanocaldococcaceae or Methanococcaceae. In contrast, bacterial IPLs dominate IPL distributions from LSHF, suggesting that bacteria are more predominant at LSHF than at RHF. Bacterial Diacyl glycerol ( DAG) IPLs containing phosphocholine, phosphoethanolamine or phosphoglycerol head groups were identified at both vent fields. In some vent deposits from LSHF ornithine lipids and IPLs containing phosphoaminopentanetetrol head groups were also observed. By comparison with previously characterized bacterial communities at the sites, it is likely the DAG- IPLs observed derive from Epsilon- and Gammaproteobacteria. Variation in the relative amounts of archaeal versus bacterial IPLs appears to indicate differences in the microbial community between vent sites. Overall, IPL distributions appear to be consistent with gene-based surveys. [ABSTRACT FROM AUTHOR]

A novel proxy for continental mean annual air temperature (MAAT) and soil-pH, the MBT/CBT-paleothermometer, is based on the temperature (T) and pH-dependent distribution of specific bacterial membrane lipids (branched glycerol dialkyl glycerol tetraethers - GDGTs) in soil organic matter. Here, we tested the applicability of the MBT/CBT-paleothermometer to sediments from Lake Cadagno, a small high-alpine lake in southern Switzerland with a small catchment of 2.4 km⊃2. We analysed the distribution of bacterial GDGTs in catchment soils and in a radiocarbon-dated sediment core from the centre of the lake, covering the entire Holocene. The composition of bacterial GDGTs in soils are almost identical to that in the lake's surface sediments, indicating a common origin of the lipids. Consequently, their transfer from the soils into the sediment record is undisturbed, apparently without any significant alteration of their distribution through in situ production or early diagenesis of branched GDGTs. The MBT/CBT-inferred MAAT-estimates from soils and surface sediments are in good agreement with instrumental values for the Lake Cadagno region (~0.5 °C). Moreover, downcore MBT/CBT-derived MAAT-estimates match in timing and magnitude other proxy-based T -reconstructions from nearby locations for the last two millennia. Major climate anomalies recorded by the MBT/CBT-paleothermometer are, for instance, the Little Ice Age (~14th to 19th century) and the Medieval Warm Period (~10th to 14th century). Together, our observations confirm the applicability of the MBT/CBT-paleothermometer to Lake Cadagno sediments. Consistent with other T-records from both the Alps and from the subpolar NE-Atlantic, our lacustrine paleotemperature record indicates Holocene MAAT-variations with an apparent cyclicity of ~2 kyr. The good temporal match of the warm periods determined for the S-Alpine region with NW-European winter precipitation strength implies a strong and far-reaching influence of the North Atlantic Oscillation on continental European Holocene T-variations. [ABSTRACT FROM AUTHOR]

Although most gastric cancers occur in elderly patients, a substantial number of cases of this common disease occur in young patients. Gastric cancer is a heterogeneous disease at the genomic level and different patterns of DNA copy number alterations are associated with different clinical behaviour. The aim of the present study was to explore differences in DNA copy number alterations in relation to age of onset of gastric cancer. DNA isolated from 46 paraffin-embedded gastric cancer tissue samples from 17 patients less than 50 years of age [median 43 (21-49) years] and 29 patients greater than or equal to 70 years of age [median 75 (70-83) years] was analysed by genome-wide microarray comparative genomic hybridization (array CGH) using an array of 5000 BAC clones. Patterns of DNA copy number aberrations were analysed by hierarchical cluster analysis of the mode-normalized and smoothed log2 ratios of tumour to normal reference fluorescence signal intensities using TMEV software, after which cluster membership was correlated with age group. In addition, supervised analysis was performed using CGH Multi-array. Hierarchical cluster analysis of the array CGH data revealed three clusters with different genomic profiles that correlated significantly with age ( p = 0.006). Cluster 1 mainly contained young patients, while elderly patients were divided over clusters 2 and 3. Chromosome regions 11q23.3 and 19p13.3 contributed most to age-related differences in tumour profiles. Gastric cancers of young and old patients belong to groups with different genomic profiles, which likely reflect different pathogenic mechanisms of the disease. Copyright © 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

We developed a sensitive sequencing approach that simultaneously profiles microsatellite instability, chromosomal instability, and subclonal structure in cancer. We assessed diverse repeat motifs across 225 microsatellites on colorectal carcinomas. Our study identified elevated alterations at both selected tetranucleotide and conventional mononucleotide repeats. Many colorectal carcinomas had a mix of genomic instability states that are normally considered exclusive. An MSH3 mutation may have contributed to the mixed states. Increased copy number of chromosome arm 8q was most prevalent among tumors with microsatellite instability, including a case of translocation involving 8q. Subclonal analysis identified co-occurring driver mutations previously known to be exclusive., (© 2021. The Author(s).)

Background: Rare single nucleotide variants play an important role in genetic diversity and heterogeneity of specific human disease. For example, an individual clinical sample can harbor rare mutations at minor frequencies. Genetic diversity within an individual clinical sample is oftentimes reflected in rare mutations. Therefore, detecting rare variants prior to treatment may prove to be a useful predictor for therapeutic response. Current rare variant detection algorithms using next generation DNA sequencing are limited by inherent sequencing error rate and platform availability., Findings: Here we describe an optimized implementation of a rare variant detection algorithm called RVD for use in targeted gene resequencing. RVD is available both as a command-line program and for use in MATLAB and estimates context-specific error using a beta-binomial model to call variants with minor allele frequency (MAF) as low as 0.1%. We show that RVD accepts standard BAM formatted sequence files. We tested RVD analysis on multiple Illumina sequencing platforms, among the most widely used DNA sequencing platforms., Conclusions: RVD meets a growing need for highly sensitive and specific tools for variant detection. To demonstrate the usefulness of RVD, we carried out a thorough analysis of the software's performance on synthetic and clinical virus samples sequenced on both an Illumina GAIIx and a MiSeq. We expect RVD can improve understanding the genetics and treatment of common viral diseases including influenza. RVD is available at the following URL:http://dna-discovery.stanford.edu/software/rvd/.

We have performed the first extensive profiling of Epstein-Barr virus (EBV) miRNAs on in vivo derived normal and neoplastic infected tissues. We describe a unique pattern of viral miRNA expression by normal infected cells in vivo expressing restricted viral latency programs (germinal center: Latency II and memory B: Latency I/0). This includes the complete absence of 15 of the 34 miRNAs profiled. These consist of 12 BART miRNAs (including approximately half of Cluster 2) and 3 of the 4 BHRF1 miRNAs. All but 2 of these absent miRNAs become expressed during EBV driven growth (Latency III). Furthermore, EBV driven growth is accompanied by a 5-10 fold down regulation in the level of the BART miRNAs expressed in germinal center and memory B cells. Therefore, Latency III also expresses a unique pattern of viral miRNAs. We refer to the miRNAs that are specifically expressed in EBV driven growth as the Latency III associated miRNAs. In EBV associated tumors that employ Latency I or II (Burkitt's lymphoma, Hodgkin's disease, nasopharyngeal carcinoma and gastric carcinoma), the Latency III associated BART but not BHRF1 miRNAs are up regulated. Thus BART miRNA expression is deregulated in the EBV associated tumors. This is the first demonstration that Latency III specific genes (the Latency III associated BARTs) can be expressed in these tumors. The EBV associated tumors demonstrate very similar patterns of miRNA expression yet were readily distinguished when the expression data were analyzed either by heat-map/clustering or principal component analysis. Systematic analysis revealed that the information distinguishing the tumor types was redundant and distributed across all the miRNAs. This resembles "secret sharing" algorithms where information can be distributed among a large number of recipients in such a way that any combination of a small number of recipients is able to understand the message. Biologically, this may be a consequence of functional redundancy between the miRNAs.

Array-based comparative genomic hybridization is a high resolution method for measuring chromosomal copy number changes. Here we present a validated protocol using in-house spotted oligonucleotide libraries for array comparative genomic hybridization (CGH). This oligo array CGH platform yields reproducible results and is capable of detecting single copy gains, multi-copy amplifications as well as homozygous and heterozygous deletions as small as 100 kb with high resolution. A human oligonucleotide library was printed on amine binding slides. Arrays were hybridized using a hybstation and analysed using BlueFuse feature extraction software, with >95% of spots passing quality control. The protocol allows as little as 300 ng of input DNA and a 90% reduction of Cot-1 DNA without compromising quality. High quality results have also been obtained with DNA from archival tissue. Finally, in addition to human oligo arrays, we have applied the protocol successfully to mouse oligo arrays. We believe that this oligo-based platform using 'off-the-shelf' oligo libraries provides an easy accessible alternative to BAC arrays for CGH, which is cost-effective, available at high resolution and easily implemented for any sequenced organism without compromising the quality of the results.

Isoprenoid glycerol dialkyl glycerol tetraethers (GDGTs) and branched glycerol dialkyl diethers are main membrane constituents of cultured hyperthermophilic archaea and eubacteria, respectively, and are found in environments with temperatures >60 degrees C. Recently, we developed a new technique for the analysis of intact core tetraether lipids in cell material and sediments. The application of this technique to recent sediments shows that known and newly identified isoprenoid and branched GDGTs are widespread in low-temperature environments (<20 degrees C) and are structurally far more diverse than previously thought. Their distribution indicates the ubiquitous environmental presence of as yet uncultivated, nonthermophilic organisms that may have independently evolved from hyperthermophilic archaea and eubacteria. The structures of some of the new GDGTs point to the hybridization of both typical archaeal and eubacterial biosynthetic pathways in single organisms.

Four previously unknown n-C25 and n-C27 heptaenes of the marine diatom Rhizosolenia setigera were isolated and identified using NMR spectroscopy. They possess six methylene interrupted (Z)-double bonds starting at C-3 and an additional terminal or n-2 (Z)-double bond. Structural and stable carbon isotopic evidence suggests that these polyenes are biosynthesized by chain elongation of the C22:6n-3 fatty acid, followed by decarboxylation and introduction of double bonds at specific positions.

A method combining normal phase high performance liquid chromatography (HPLC) with positive ion atmospheric pressure chemical ionization mass spectrometry (APCI-MS) was developed for the analysis of intact glycerol dialkyl glycerol tetraethers (GDGTs) in archaeal cell material and sediments. All GDGTs previously reported to occur in the thermophilic archaeon Sulfolobus solfataricus could be identified based on their mass spectra and retention time. Positive ion mass spectra consisted of abundant protonated molecules and fragment ions corresponding to loss of water and the glycerol moiety. In addition, two novel GDGTs representing alternative combinations of biphytanyl moieties were observed. Using this method, the tetraethers present in the thermophilic archaeon Metallosphaera sedula and two sediment samples were characterized. This rapid method will greatly contribute to the establishment of the sedimentary record of these compounds and increase our understanding of archaea and their occurrence in widely different environments.

Carcinogenic and mutagenic heterocyclic aromatic amines (HAA) are natural products often present at ng/g levels in muscle meats when they are cooked at temperatures over 150 degrees C. Using solid-phase extraction and high performance liquid chromatography (HPLC) with photodiode array UV detection, samples were analyzed for the following heterocyclic amines: DiMeIQx (2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline); IQ (2-amino-3-methylimidazo[4,5-f]quinoline); MelQx (2- amino-3,8-dimethylimidazo[4,5-f]quinoxaline); and PhIP (2-amino-1-methyl- 6-phenylimidazo[4,5-b]pyridine). Quality control samples, analyzed periodically over two years in a blind study, show relative standard deviations ranging from 22 to 38% for the compounds found, variations typical for analysis at ng/g levels. Amounts range from undetectable levels (less than 0.1 ng/g) to hundreds of ng/g of PhIP for frying or grilling at high meat surface temperatures. Beef, chicken, pork and lamb can all have greater than 10 ng/g of PhIP. Ground chicken breast meat has lower amounts of heterocyclic amines than intact muscle pieces of the same size cooked identically. Restaurant prepared samples that we analyzed contained undetectable levels up to 14 ng/g total heterocyclic amines for a beef steak sample. Not extracted with the above method are related mutagenic heterocyclic amines, which have been reported in cooked foods in our laboratory and others. Method development using ion exchange on an SCX solid-phase extraction cartridge shows promise in providing a method for the quantitation of these mutagenic dimethyl-, trimethyl- and furo-imidazopyridines where a practical analysis method is needed.

Fumonisins (FBs) are a family of mycotoxins produced by Fusarium moniliforme and F. proliferatum, predominant corn pathogens, and are found in most corn-containing foods. The FBs are heat stable, resistant to ammoniation, and unlike most mycotoxins, are water-soluble. The levels in corn and corn-containing foods will be presented ranging from < 20 ppb to > 2 ppm. Washing of contaminated FB-corn with water did not reduce the measured FB levels of significantly. The traditional processing step to make tortilla flour, nixtamalization [Ca(OH)2 cooking] to produce masa, reduced FB levels but produced hydrolyzed FB which was almost as toxic as FB. Retorting sweet corn in brine apparently produced hydrolyzed FB. Fermentation of corn to ethanol did not alter FB levels but distillation yielded FB-free ethanol. Attempts to enzymatically modify FB with several enzymes were unsuccessful. Reactions between FB and reducing sugars (glucose or fructose) to produce Schiff's bases yielded products that were not toxic. The effects of these processing treatments must be evaluated both chemically and biologically.

Groups of 5-w-old F344/N female rats were fed a semipurified diet for 13 w with or without 20 mg fumonisin B1/kg provided from an aqueous extract of Fusarium moniliforme-corn culture. After 1 w, a single dose of 30 mg diethylnitrosamine (DEN)/kg was given orally. Twelve weeks later, the presence of placental glutathione S-transferase-positive (PGST-[+]) hepatocytes were immunohistochemically quantified. Rats given DEN and the FB1-containing diet for 1 or 13 w developed 4-fold more PGST-[+] hepatocytes than rats given DEN alone. In a second study, male and female F344/N rats were fed 20 mg purified FB1/kg diet or F proliferatum-corn culture material containing 20 mg FB1/kg diet for 1 w before DEN treatment. One week after DEN treatment, male rats fed the F proliferatum-corn culture material had significantly fewer PGST-[+] hepatocytes than those fed DEN with or without purified FB1. At 9 w after DEN treatment, PGST-[+] cells in female rats given DEN and fed F proliferatum-corn culture material were more persistent than in rats given DEN alone. Males given DEN and fed FB1 or F proliferatum culture material had significantly fewer PGST-[+] hepatocytes than males given DEN alone. These results suggest that F moniliforme and F proliferatum components are cocarcinogens in females. In males, however, FB1 and unidentified F proliferatum components reduced the persistence of DEN-initiated preneoplastic hepatocytes.

Groups of 5-6 pregnant F344/N rats were dosed (po) from d 8 to 12 of gestation with 30 or 60 mg purified fumonisin B1 (FB1)/kg body weight, or with a fat-soluble extract of Fusarium proliferatum/corn culture derived from an amount of corn culture that would provide approximately 60 mg FB1/kg. Control rats were dosed with water or corn oil. Food intake was monitored daily during dosing. Fetal bone development was examined after staining with alizarin red, whereas internal organ development was examined in hematoxylin and eosin-stained tissue sections. Although group differences in maternal body weight were not statistically significant, weight was 6% less in dams dosed with 60 mg FB1/kg compared with the control group (p < 0.12). Relative litter weight was significantly suppressed by 60 mg FB1/kg. Ossification of the sternebrae and vertebral bodies was significantly impaired by FB1 treatment. Litters from mothers treated with a fat-soluble extract of F proliferatum/corn culture did not have suppression of weight or impairment of bone development. Fumonisin B1 is fetotoxic to rats by suppressing growth and fetal bone development.

A new heterocyclic amine mutagen was isolated from a dry-heated reaction of the natural meat components creatine, glutamic acid and glucose. Heating creatine and glutamic acid alone had only one seventh of the Ames/Salmonella mutagenic activity of the glucose, creatine and glutamic acid mixture. The major mutagenic compound was purified by HPLC using the Ames/Salmonella test to guide the purification. The mutagen has a molecular weight of 244 and a composition of C12H12N4O2 as determined by high-resolution mass spectrometry. NMR and IR spectral data suggest the structure is a 2,6-diamino-3,4-dimethyl-7-oxo-pyrano[4,3-g]benzimidazole. Mutagenic activity in strains TA1538, TA98 and TA100, was approximately 7000, 5200, and 550 revertants per microgram, respectively. The formation of this mutagen from natural meat components suggests that it may be present in cooked food. The preferential formation of this mutagen with glucose shows that glucose can be important in dry-heated mutagen-forming reactions.