4D Visualization of replication foci in mammalian cells corresponding to individual replicons.

Since the pioneering proposal of the replicon model of DNA replication 50 years ago, the predicted replicons have not been identified and quantified at the cellular level. Here, we combine conventional and super-resolution microscopy of replication sites in live and fixed cells with computational image analysis. We complement these data with genome size measurements, comprehensive analysis of S-phase dynamics and quantification of replication fork speed and replicon size in human and mouse cells. These multidimensional analyses demonstrate that replication foci (RFi) in three-dimensional (3D) preserved somatic mammalian cells can be optically resolved down to single replicons throughout S-phase. This challenges the conventional interpretation of nuclear RFi as replication factories, that is, the complex entities that process multiple clustered replicons. Accordingly, 3D genome organization and duplication can be now followed within the chromatin context at the level of individual replicons.

3D replicon distributions arise from stochastic initiation and domino-like DNA replication progression.

DNA replication dynamics in cells from higher eukaryotes follows very complex but highly efficient mechanisms. However, the principles behind initiation of potential replication origins and emergence of typical patterns of nuclear replication sites remain unclear. Here, we propose a comprehensive model of DNA replication in human cells that is based on stochastic, proximity-induced replication initiation. Critical model features are: spontaneous stochastic firing of individual origins in euchromatin and facultative heterochromatin, inhibition of firing at distances below the size of chromatin loops and a domino-like effect by which replication forks induce firing of nearby origins. The model reproduces the empirical temporal and chromatin-related properties of DNA replication in human cells. We advance the one-dimensional DNA replication model to a spatial model by taking into account chromatin folding in the nucleus, and we are able to reproduce the spatial and temporal characteristics of the replication foci distribution throughout S-phase.

Epigenetic modifications in sex heterochromatin of vole rodents.

The genome of some vole rodents contains large blocks of heterochromatin coupled to the sex chromosomes. While the DNA content of these heterochromatic blocks has been extensively analyzed, little is known about the epigenetic modifications controlling their structure and dynamics. To better understand its organization and functions within the nucleus, we have compared the distribution pattern of several epigenetic marks in cells from two species, Microtus agrestis and Microtus cabrerae. We first could show that the heterochromatic blocks are identifiable within the nuclei due to their AT enrichment detectable by DAPI staining. By immunostaining analyses, we demonstrated that enrichment in H3K9me3 and HP1, depletion of DNA methylation as well as H4K8ac and H3K4me2, are major conserved epigenetic features of this heterochromatin in both sex chromosomes. Furthermore, we provide evidence of transcriptional activity for some repeated DNAs in cultivated cells. These transcripts are partially polyadenylated and their levels are not altered during mitotic arrest. In summary, we show here that enrichment in H3K9me3 and HP1, DNA demethylation, and transcriptional activity are major epigenetic features of sex heterochromatin in vole rodents.

New image colocalization coefficient for fluorescence microscopy to quantify (bio-)molecular interactions.

The spatial relationship, or degree of colocalization, between two or more types of molecules in live cells is commonly detected using fluorescence microscopy. This spatial distribution can be used to estimate the interaction between fluorescently labelled molecules. These interactions are usually quantified by analysing the correlation and/or the overlap between images, using the Pearson's and Manders' coefficients, respectively. However, the correlation and overlap coefficients are parameters not designed to quantify molecular interactions. Here we propose a new colocalization coefficient specifically designed to quantify the interactions between molecules. In well-defined thermodynamic ensembles, this coefficient can in principle be used to calculate relevant statistical thermodynamic quantities such as binding free energies.

[Fibrillary glomerulonephritis of the nodular glomerulosclerosis variety]. / Glomerulonefritis fibrilar variedad glomeruloesclerosis nodular.

We report a 63 years old female patient presenting with progressive edema, alopecia and pallor. Laboratory showed a proteinuria of 1.9 g/24 h, microhematuria, a serum creatinine of 3.1 mg/dl, erythrocyte sedimentation rate of 133 mm/h and antinuclear antibodies of 1/40 with a homogeneous pattern. No extrarenal disease was demonstrated and a kidney biopsy, performed 18 months later, showed a fibrillary glomerulonephritis, nodular sclerosis variety, with 20 nm phi fibrillae, immune and kappa and lambda light chain deposits and negative Congo red stain. The patient died 30 months later due to a respiratory infection. Fibrillary glomerulonephritis is an infrequent form of mesangiocapillary glomerulonephritis not associated to plasma cell dyscrasia. It leads to terminal renal failure and it recurs in transplanted kidneys.

Effects of postnatal age and diet on the fatty acid composition of plasma lipid fractions in preterm infants.

Diet and postnatal age effect the fatty acid composition of plasma and tissue lipids. This work was designed as a transversal study to evaluate the changes in the fatty acid composition of plasma phospholipids, cholesteryl esters, triglycerides and free fatty acids in preterm infants (28-35 weeks gestational age), fed human milk (HM) and milk formula (MF) from birth to 1 month of life. Sixteen blood samples were obtained from cord, and 19 at 6-8 h after birth, 14 at 1 week and 9 at 4 weeks from HM-fed infants and 18 at 1 week and 14 at 4 weeks from MF-fed ones. Groups had similar mean birth weight, gestational age and sex ratio. The MF provided 69 kcal/dl and contained 16% of linoleic acid and 1.3% of alpha-linolenic acid on the total fat. Plasma lipid fractions were extracted and separated by thin-layer chromatography and fatty acid methyl esters were quantitated by gas liquid chromatography. In plasma phospholipids, linoleic acid (18:2 omega 6) continuously increased from birth to 1 month of age, but no changes were seen as related to type of diet; polyunsaturated fatty acids greater than 18 carbon atoms of both the omega 6 and omega 3 series (PUFA omega 6 greater than 18 C and omega 3 greater than 18 C) dropped from birth to 1 week and continued to decrease in MF-fed infants until 1 month; eicosatrienoic (20:3 omega 6), arachidonic (20:4 omega 6) and docosahexaenoic (22:6 omega 3) were the fatty acids implicated. In cholesteryl esters palmitoleic (16:1 omega 7) and oleic (18:1 omega 9) acids decreased from birth to 1 month and linoleic acid increased and arachidonic acid dropped, especially in MF fed infants. In triglycerides, palmitic, palmitoleic and stearic acid (18:0) decreased during the first month of life; oleic acid remained constant and linoleic acid increased in all infants, but arachidonic acid decreased only in those fed formula. Free fatty acids showed a similar behavior in fatty acids and in plasma triglycerides. Preterm neonates seem to have special requirements of long-chain PUFA and adapted MF should contain these fatty acids in similar amounts to those of HM to allow the maintenance of an adequate tissue structure and physiology.

Movement patterns of invertebrates in temporary and permanent streams.

Although it has been shown that invertebrates recolonize reflooded temporary streams from permanent refuges, e.g., the hyporheic zone, it has not been shown that they actively move into these refuges as streams dry. Substrate filled cages and drift nets were used to monitor invertebrate movement in two temporary streams and a permanent stream prior to and during drying to determine whether invertebrates leave drying riffles and enter flooded riffles. Invertebrate movement was essentially unidirectional in the permanent stream with downstream drift and with-in-substrate downstream movement dominating. In the temporary stream, movement vertically downward toward the hyporheic zone and upstream movement substantially contributed to a departure from a unidirectional pattern. In addition, prior to stream drying the relative colonization rate was higher and drift rate was lower in the temporary streams than in the permanent stream. During drying of the temporary stream, upstream movement continued to dominate but hyporheic movement was unimportant. Further, the upstream movement did not occur at the end of the riffle where it would lead to migration into non-drying riffles. Thus, even though movement patterns were different in permanent and temporary streams the pattern observed during stream drying would result in the concentration and subsequent death of invertebrates in drying riffles. This observation demonstrates that movement patterns of stream invertebrates do not necessarily result in behavioral avoidance of a dry period of temporary fiffles.

Effects of dietary nucleotides on the fatty acid composition of erythrocyte membrane lipids in term infants.

Human milk contains relatively high amounts of acid-soluble nucleotides, mainly CMP, AMP, UMP, IMP, and UDP sugars, and lacks orotate. On the contrary, cow's milk and adapted formulas contain high amounts of orotate and very low amounts of CMP and AMP. Nucleotides may be modulators of metabolic functions in gut and liver. To evaluate the possible role of dietary nucleotides in newborn fatty acid metabolism, we studied the polyunsaturated fatty acid (PUFA) composition of erythrocyte membrane phospholipids in 58 term infants at 30 days of age. Twenty of them were fed human milk (HM), 19 an adapted formula (MF), and 19 with the same formula supplemented with nucleotides (NMF) in an amount similar to that present in HM. Relative content of omega 6 and 3 PUFA greater than 18 carbon atoms was significantly reduced especially in phosphatidylethanolamine and phosphatidylserine for infants fed regular MF compared with those fed HM or NMF. Unsaturation index of red blood cell phospholipids showed a similar effect. These results suggest that dietary nucleotides play a role in the in vivo desaturation and elongation of essential fatty acids to long chain PUFA during early life for the human newborn.