Heterogeneous melting near the Thwaites Glacier grounding line.

Thwaites Glacier represents 15% of the ice discharge from the West Antarctic Ice Sheet and influences a wider catchment1-3. Because it is grounded below sea level4,5, Thwaites Glacier is thought to be susceptible to runaway retreat triggered at the grounding line (GL) at which the glacier reaches the ocean6,7. Recent ice-flow acceleration2,8 and retreat of the ice front8-10 and GL11,12 indicate that ice loss will continue. The relative impacts of mechanisms underlying recent retreat are however uncertain. Here we show sustained GL retreat from at least 2011 to 2020 and resolve mechanisms of ice-shelf melt at the submetre scale. Our conclusions are based on observations of the Thwaites Eastern Ice Shelf (TEIS) from an underwater vehicle, extending from the GL to 3 km oceanward and from the ice-ocean interface to the sea floor. These observations show a rough ice base above a sea floor sloping upward towards the GL and an ocean cavity in which the warmest water exceeds 2 °C above freezing. Data closest to the ice base show that enhanced melting occurs along sloped surfaces that initiate near the GL and evolve into steep-sided terraces. This pronounced melting along steep ice faces, including in crevasses, produces stratification that suppresses melt along flat interfaces. These data imply that slope-dependent melting sculpts the ice base and acts as an important response to ocean warming.

Recent advances in circulating nucleic acids in oncology.

Circulating cell-free DNA (cfDNA) is one of the fastest growing and most exciting areas in oncology in recent years. Its potential clinical uses cover now each phase of cancer patient management care (predictive information, detection of the minimal residual disease, early detection of resistance, treatment monitoring, recurrence surveillance, and cancer early detection/screening). This review relates the recent advances in the application of circulating DNA or RNA in oncology building on unpublished or initial findings/work presented at the 10th international symposium on circulating nucleic acids in plasma and serum held in Montpellier from the 20th to the 22nd of September 2017. This year, presenters revealed their latest data and crucial observations notably in relation to (i) the circulating cell-free (cfDNA) structure and implications regarding their optimal detection; (ii) their role in the metastatic or immunological processes; (iii) evaluation of miRNA panels for cancer patient follow up; (iv) the detection of the minimal residual disease; (v) the evaluation of a screening tests for cancer using cfDNA analysis; and (vi) elements of preanalytical guidelines. This work reviews the recent progresses in the field brought to light in the meeting, as well as in the most important reports from the literature, past and present. It proposes a broader picture of the basic research and its potential, and of the implementation and current challenges in the use of circulating nucleic acids in oncology.

Origins, structures, and functions of circulating DNA in oncology.

While various clinical applications especially in oncology are now in progress such as diagnosis, prognosis, therapy monitoring, or patient follow-up, the determination of structural characteristics of cell-free circulating DNA (cirDNA) are still being researched. Nevertheless, some specific structures have been identified and cirDNA has been shown to be composed of many "kinds." This structural description goes hand-in-hand with the mechanisms of its origins such as apoptosis, necrosis, active release, phagocytosis, and exocytose. There are multiple structural forms of cirDNA depending upon the mechanism of release: particulate structures (exosomes, microparticles, apoptotic bodies) or macromolecular structures (nucleosomes, virtosomes/proteolipidonucleic acid complexes, DNA traps, links with serum proteins or to the cell-free membrane parts). In addition, cirDNA concerns both nuclear and/or mitochondrial DNA with both species exhibiting different structural characteristics that potentially reveal different forms of biological stability or diagnostic significance. This review focuses on the origins, structures and functional aspects that are paradoxically less well described in the literature while numerous reviews are directed to the clinical application of cirDNA. Differentiation of the various structures and better knowledge of the fate of cirDNA would considerably expand the diagnostic power of cirDNA analysis especially with regard to the patient follow-up enlarging the scope of personalized medicine. A better understanding of the subsequent fate of cirDNA would also help in deciphering its functional aspects such as their capacity for either genometastasis or their pro-inflammatory and immunological effects.

Microsatellite alterations in serum DNA of head and neck cancer patients.

Microsatellite DNA alterations are an integral part of neoplastic progression and are valuable as clonal markers for the detection of human cancers. Moreover, recent evidence suggests that senescent tumor cells may release DNA into the circulation, which is subsequently carried by and therefore enriched in the serum and plasma. We tested 21 patients with primary head and neck squamous cell carcinoma (HNSCC) by polymerase chain reaction (PCR)-based microsatellite analysis of DNA from lymphocytes and paired serum samples. Patients were scored for alterations as defined by the presence of new alleles (shifts) or loss of heterozygosity (LOH) in serum at each of 12 markers and then compared with primary tumor DNA. Six out of 21 patients (29%) were found to have one or more microsatellite alterations in serum precisely matching those in the primary tumors. All six patients had advanced disease (stage III or IV); five of these patients had nodal metastases, three later developed distant metastases, and four died of disease. Microsatellite analysis of serum represents a novel method for the detection of circulating tumor cell DNA. If these results are confirmed in larger studies, microsatellite markers may be useful in assessing tumor burden in cancer patients.

Microsatellite alterations in plasma DNA of small cell lung cancer patients.

Microsatellite instability is an important characteristic of many tumor types especially those associated with hereditary non-polyposis colorectal carcinoma (HNPCC) syndrome. Microsatellite alterations in 50% of primary small cell lung carcinoma (SCLC) have been found. These alterations were also found in the sputum. Because neoplastic characteristics such as decreased strand stability9 and ras mutations have been found in the plasma DNA of cancer patients, we looked for microsatellite alterations in the plasma of SCLC patients. A microsatellite alteration was present in 16 out of 21 (76%) SCLC tumors and in 15 out of 21 (71%) plasma samples. In one case, the alteration was present only in the plasma DNA. If confirmed in larger studies, microsatellite analysis of plasma DNA might constitute a new tool for tumor staging, management and, possibly, detection.

Bacterial ribonucleic acid in the frog brain after a bacterial peritoneal infection.

Frogs were injected intraperitoneally with bacteria, and the RNA of the brains (which have protective barriers against the bacteria used) was extracted. Part of the RNA was bacterial RNA apparently resulting from the transcription of DNA transferred from bacteria to the brain cells.

Biochemical composition of fluids for amnioinfusion during fetoscopy.

OBJECTIVE: To evaluate which of the commercially available solutions is best suited for amnioinfusion during fetoscopy, based on resemblance with the biochemical properties of amniotic fluid. MATERIALS AND METHODS: Amniotic fluid samples from 10 pregnancies were studied. Specimens were obtained from 5 pathologic pregnancies (of which 3 were complicated by polyhydramnios) and 5 uncomplicated pregnancies. The concentrations of sodium, potassium, chloride, bicarbonate, calcium, glucose, osmolality, pH, total protein content and albumin were determined in each sample. A literature search (PubMed, Embase) was performed to identify commercially available fluids used for amnioinfusion in clinical practice. The composition of these infusion solutions was compared to the amniotic fluid samples mentioned above. RESULTS: We identified two different electrolyte solutions used in clinical practice for amnioinfusion. We identified four additional commercially available solutions that could potentially be used for amnioinfusion. Most of these infusion solutions differ considerably from midtrimester amniotic fluid samples both in electrolyte composition and pH, with the most striking difference in the latter. CONCLUSION: Lactated Ringer's solution approximates amniotic fluid the closest for both electrolyte composition and pH. This infusion solution seems to be the most suitable choice for amnioinfusion during fetoscopy.

Haplotype diversity: the link between statistical and biological association.

In the rapidly growing field of association mapping in plants, the use of (marker) haplotypes rather than single markers can be an effective way of improving detection power. Here, we highlight the information that can be obtained from deducing the historical relationships between haplotypes. The ordering of haplotype classes according to deduced historical relationships should further enhance association detection power, but can also be used to predict the genotypic and phenotypic values of unobserved germplasm.

Quantitative trait locus (QTL) isogenic recombinant analysis: a method for high-resolution mapping of QTL within a single population.

In the quest for fine mapping quantitative trait loci (QTL) at a subcentimorgan scale, several methods that involve the construction of inbred lines and the generation of large progenies of such inbred lines have been developed (Complex Trait Consortium 2003). Here we present an alternative method that significantly speeds up QTL fine mapping by using one segregating population. As a first step, a rough mapping analysis is performed on a small part of the population. Once the QTL have been mapped to a chromosomal interval by standard procedures, a large population of 1000 plants or more is analyzed with markers flanking the defined QTL to select QTL isogenic recombinants (QIRs). QIRs bear a recombination event in the QTL interval of interest, while other QTL have the same homozygous genotype. Only these QIRs are subsequently phenotyped to fine map the QTL. By focusing at an early stage on the informative individuals in the population only, the efforts in population genotyping and phenotyping are significantly reduced as compared to prior methods. The principles of this approach are demonstrated by fine mapping an erucic acid QTL of rapeseed at a subcentimorgan scale.

Spontaneous release of DNA by human blood lymphocytes as shown in an in vitro system.

Human blood lymphocytes released DNA in vitro in the absence of any stimulation. Once purified from the complex appearing in the supernatant, this DNA exhibited typical characteristics as shown by its UV absorption curve, its deoxyribose coloration, and its sensitivity to DNase. Elution patterns on hydroxyapatite columns indicated that the excreted DNA is double stranded. The released DNA was smaller than the cellular DNA; its molecular weight ranged from 3.5 x 10(5) to 3.7 x 10(6) daltons. The DNA appearing in the supernatant does not seem to be due to dead or dying cells since: (a) the same amount of DNA was found in the medium whether the incubation lasted 2 hr or as long as 16 hr; (b) cell death rate had no effect on the amount of extracellular DNA; (c) when the lymphocytes were centrifuged and placed in a new medium several times in a row, a similar amount of extracellular DNA was isolated from each of the successive supernatants, whereas if, after centrifugation, the lymphocytes were put back in their original medium, no increase in the amount of extracellular DNA was observed, suggesting an active regulatory mechanism independent of a mechanical effect; (d) it took more than 1 hr for extracellular DNA to reach its maximum concentration, a fact that also argues against a mechanical effect; (e) the specific activity of the released DNA was different from that of the cellular DNA, depending on the time of labeling; and (f) the cells that had excreted DNA kept their functional integrity, as shown by their fully maintained capacity to increase DNA synthesis after stimulation. The extracellular DNA hybridized specifically with cellular DNA. The hybridization curves indicate that the DNA excreted is highly complex, and they suggest that it is composed of part of the newly synthesized DNA. The higher specific activity of the released DNA, compared with that of the cellular DNA after a long labeling period, can be explained only by a preferential release of the newly synthesized DNA.

Spontaneous extracellular synthesis of DNA released by human blood lymphocytes.

Human lymphocytes were shown to release, in vitro and in the absence of any stimulation, a complex containing DNA. It has also been reported that the release process is unrelated to cell death and is regulated by a homeostatic mechanism. Some properties of the extracellular DNA were investigated. When a phosphorylated precursor was added to the cell-free supernatant, the DNA recovered from the medium was labeled. Evidence that DNA lebeling represented true precursor incorporation and not simple attachment was obtained from nearest neighbor analysis data. When [alpha-32P]thymidine triphosphate was added to the supernatant and the labeled DNA was completely hydrolyzed to 3'-deoxyribonucleotides, radioactivity was found in all four nucleotides. Although the exact kind of synthesis cannot be determined at this stage, the possibility of a terminal transferase system in which the enzyme would merely add a nucleotide at the end of the chain was eliminated since comparative digestion with DNase and venom phosphodiesterase showed that labeling was located along the whole length of the chain. Precursor incorporation into the DNA was inhibited by DNase, RNase, Pronase, and actinomycin D. This extracellular synthesis was not affected by cell death rate. The renaturation curve of the extracellular [3H]DNA synthesized in the cell-free medium showed a lack of gene reiteration suggesting a preferential synthesis of unique sequences.

Presence of RNA in the nucleoprotein complex spontaneously released by human lymphocytes and frog auricles in culture.

Cell systems as different as normal human blood lymphocytes and frog auricles release spontaneously a nucleoprotein complex in their culture medium. This release seems to be an active mechanism that is unrelated to cell death. The presence of RNA in this complex is demonstrated. The amount of extracellular RNA is regulated by the same homeostatic mechanism that has previously been shown to govern DNA release in the same cell systems. This extracellular RNA is linked by hydrogen bonds to the extracellular DNA and cannot be extracted by a usual phenol procedure, due perhaps to the presence of a glycoprotein. Further purifications by chloroform, sodium perchlorate, and hydroxyapatite are necessary to obtain an RNA molecule that is acid precipitable, RNase and KOH sensitive, and orcinol positive. The extracellular RNA sediments between 2.5 and 4S and is not a transfer RNA. It is more highly methylated than the 28S, 18S, and 4 to 5S cellular RNA. It activates DNA synthesis in vitro.

Microsatellite analysis of plasma DNA from patients with clear cell renal carcinoma.

Deletions of DNA sequences on chromosome 3p [loss of heterozygosity (LOH)] are characteristic of clear cell renal carcinoma, which accounts for about 80% of all renal malignancies. Comparing tumor DNA to DNA from normal cells, LOH analysis of microsatellite sequences has aided in molecular diagnosis of renal carcinoma. Because clinically useful tumor markers do not exist for this cancer entity, the aim of the present study was to detect chromosome 3p microsatellite alterations (LOH and microsatellite instability) in plasma DNA from patients with clear cell renal carcinoma. Four chromosome 3p microsatellites (D3S1307, D3S1560, D3S1289, and D3S1300) were amplified by fluorescent PCR using DNA isolated from normal blood cells and plasma of 40 patients. Corresponding tumor DNA was available from 21 patients. Analyzing PCR products on an automated DNA sequencer, we found LOH in at least one locus in 25 plasma samples (63%), and 14 plasma samples (35%) exhibited LOH at more than one locus. Microsatellite instability of plasma DNA was detectable in one patient (3%). No significant association of advanced (>T2N0M0) tumor stages with LOH in plasma DNA could be demonstrated. If present, modifications of plasma DNA and tumor DNA were identical. No alterations of plasma DNA were found in healthy controls. Analysis of plasma DNA from patients with clear cell renal carcinoma reveals tumor-specific microsatellite alterations and may therefore have diagnostic potential as a molecular tumor marker.

Telomerase RNA as a detection marker in the serum of breast cancer patients.

Tumor-derived circulating DNA has been found in the plasma of cancer patients. Alterations include decreased strand stability, mutations of oncogenes or of tumor suppressor genes, microsatellite alterations, and hypermethylation of several genes. RNA has also been found circulating in the plasma of normal subjects and cancer patients. Tyrosinase mRNA has been extracted from the serum of melanoma patients and subjected to RT-PCR. Moreover, the presence of cell-free EBV-associated RNA has been reported in the plasma of patients with nasopharyngeal carcinoma. Human telomerase comprises two RNA subunits, telomerase RNA template (hTR) and its catalytic component, telomerase reverse transcriptase protein (hTERT). Expression of these subunits correlates with telomerase activity. Using RT-PCR, we investigated whether these RNA subunits were present in the serum of 18 patients with breast cancer, 2 patients with benign breast disease, and 21 normal subjects. The presence of amplifiable RNA was confirmed in all tissue and serum samples using RT-PCR of glyceraldehyde-3-phosphate dehydrogenase RNA. hTR was found in 17 of 18 tumors (94%) and 5 of 18 serum samples (28%). hTERT was also detected in 17 of 18 tumors (94%) and in 4 of 16 available serum samples (25%). hTR and hTERT were undetectable in tissues and sera taken from 2 patients with benign disease and in the sera of 21 normal subjects. We conclude that RNA is detectable in the serum of breast cancer patients and that tumor-derived mRNA can be extracted and amplified using RT-PCR, even in patients with localized disease. This may have implications for cancer diagnosis and follow-up in the future.

A prospective study of K-ras mutations in the plasma of pancreatic cancer patients.

K-ras mutations are frequently found in primary pancreatic adenocarcinomas. In this prospective study, we looked for K-ras mutations in the plasma of patients with pancreatic cancer. We isolated plasma DNA from 21 pancreatic cancer patients using a simple and rapid extraction technique and detected K-ras alterations with a PCR assay and subsequent product sequencing. Patients were followed up to determine their clinical outcome. We found K-ras mutations in the plasma of 17 patients (81%). In cases in which both plasma and pancreatic tissue were available, DNA mutations were similar in corresponding plasma and tissue samples. Plasma DNA alterations were found 5-14 months before clinical diagnosis in four patients. Mutant DNA was not found in the plasma of two patients with chronic pancreatitis or in five healthy controls. Our results indicate that K-ras mutations are often found in DNA isolated from the plasma of pancreatic cancer patients and that a noninvasive plasma-based assay may provide qualitative diagnostic information to clinicians in the future. Larger studies are required to further assess the relevance of our findings to clinical practice.

Detecting tumor-related alterations in plasma or serum DNA of patients diagnosed with breast cancer.

Chromosomal abnormalities are associated with the development of breast cancer, and widespread allelic loss or imbalance is frequently found in tumor tissues taken from patients with this disease. Using different markers, we studied a total of 61 patients (divided into three groups) for the presence of microsatellite instability and loss of heterozygosity (LOH) in plasma or serum DNA. Of the initial 27 patients, 35% of the tumor samples displayed LOH, whereas 15% had identical alterations in the corresponding plasma samples. In addition, the adjacent normal breast tissue of two patients also displayed LOH. In a second group of 11 patients, 45% of the tumors displayed LOH, and 27% displayed identical plasma DNA alterations; one case displayed an identical LOH in adjacent nontumor tissue. In a third series of 23 patients also studied with tetranucleotide repeats, 81% of the tumor samples displayed LOH, whereas 48% had LOH in the corresponding serum samples. The fact that small tumors (T1) of histoprognostic grade 1 or in situ carcinomas could present DNA alterations in the plasma/serum at an early stage, allied to the widely increased range of available microsatellite markers, suggests that plasma or serum DNA may become a useful diagnostic tool for early and potentially curable breast cancer.

Circulating DNA in higher organisms cancer detection brings back to life an ignored phenomenon.

The concept of circulating DNA is derived from the early transformation experiments on bacteria. We describe first experiments done with graft hybrids which could be due to circulating DNA. Work on uptake of foreign DNA by eukaryotic cells is then reported. This work led us to discover the phenomenon of transcession in plants and animals where we showed that bacterial DNA can spontaneously enter cells of eukaryotes and be transcribed. We then outline the fact that living cells spontaneously release DNA within a homeostatic mechanism. Finally we describe in more detail some experiments suggesting that an exchange of DNA between T and B lymphocytes may have immunological implications. In this work, nude mice injected with DNA excreted by antigen stimulated human T lymphocytes produced specific antibodies expressing human characteristics.

High plasma concentration of factor VIIIc is a major risk factor for venous thromboembolism.

BACKGROUND: Established risk factors, including deficiencies of protein C, protein S or antithrombin and the factor V Leiden and prothrombin mutation, are present in about one third of unselected patients with venous thromboembolism. In addition to these inherited thrombophilic defects, elevated plasma levels of factor VIIIc have been suggested to be important in the pathogenesis of (recurrent) venous thromboembolism. The objective of this study was to assess the relevance of factor VIIIc plasma concentration in consecutive patients with venous thromboembolism. METHOD: We studied the prevalence of elevated plasma levels of factor VIIIc in 65 patients with a proven single episode and in 60 matched patients with documented recurrent venous thromboembolism. The reference group consisted of 60 age- and sex-matched patients who were referred for suspected venous thromboembolism, which was refuted by objective testing and long-term clinical follow-up. To minimalize the influence of the acute phase, blood was obtained at least 6 months after the thromboembolic event and results were adjusted for fibrinogen and C-reactive protein. Factor VIIIc was re-determined several years after the first measurement in a subset of patients to evaluate the variability over time. To study a possible genetic cause, a family study was done. FINDINGS: In the control, single and recurrent episode group, the prevalences of plasma levels of factor VIIIc above 175 IU/dl (90th percentile of controls) were 10% (95% CI: 4 to 21%), 19% (95% CI: 10 to 30%) and 33% (95% CI: 22 to 47%), respectively. For each 10 IU/dl increment of factor VIIIc, the risk for a single and recurrent episode of venous thrombosis increased by 10% (95% CI: 0.9 to 21%) and 24% (95% CI: 11 to 38%), respectively. Both low and high plasma levels of factor VIIIc were consistent over time (R = 0.80, p = 0.01). A family study indicated a high concordance for elevated factor VIIIc plasma concentrations among first degree family members. Adjustment for fibrinogen, C-reactive protein and known thrombophilic risk factors did not change the observed association of elevated factor VIIIc with thrombosis. INTERPRETATION: Elevated plasma levels of factor VIIIc are a significant, prevalent, independent and dose-dependent risk factor for venous thromboembolism. It also predisposes to recurrent venous thromboembolism.

Developing eye in an ovarian teratoma.

A malignant teratoma of the ovary was resected in a 14-year-old girl. Within the tumor, a partially developed eye was found.