Search

Background: People with metabolically healthy (MHO) and metabolically unhealthy obesity (MUO) differ for the presence or absence of cardio-metabolic complications, respectively. Objective: Based on these differences, we are interested in deepening whether these obesity phenotypes could be linked to changes in microbiota and metabolome profiles. In this respect, the overt role of microbiota taxa composition and relative metabolic profiles is not completely understood. At this aim, biochemical and nutritional parameters, fecal microbiota, metabolome and SCFA compositions were inspected in patients with MHO and MUO under a restrictive diet regimen with a daily intake ranging from 800 to 1200 kcal. Methods: Blood, fecal samples and food questionnaires were collected from healthy controls (HC), and an obese cohort composed of both MHO and MUO patients. Most impacting biochemical/anthropometric variables from an a priori sample stratification were detected by applying a robust statistics approach useful in lowering the background noise. Bacterial taxa and volatile metabolites were assessed by qPCR and gas chromatography coupled with mass spectrometry, respectively. A targeted GC–MS analyses on SCFAs was also performed. Results: Instructed to follow a controlled and restricted daily calorie intake, MHO and MUO patients showed differences in metabolic, gut microbial and volatilome signatures. Our data revealed higher quantities of specific pro-inflammatory taxa (i.e., Desulfovibrioand Prevotellagenera) and lower quantities of Clostridium coccoides groupin MUO subset. Higher abundances in alkane, ketone, aldehyde, and indole VOC classes together with a lower amount of butanoic acid marked the faecal MUO metabolome. Conclusions: Compared to MHO, MUO subset symptom picture is featured by specific differences in gut pro-inflammatory taxa and metabolites that could have a role in the progression to metabolically unhealthy status and developing of obesity-related cardiometabolic diseases. The approach is suitable to better explain the crosstalk existing among dysmetabolism-related inflammation, nutrient intake, lifestyle, and gut dysbiosis.

Spermatozoa harbour a complex and environment-sensitive pool of small non-coding RNAs (sncRNAs)1, which influences offspring development and adult phenotypes1–7. Whether spermatozoa in the epididymis are directly susceptible to environmental cues is not fully understood8. Here we used two distinct paradigms of preconception acute high-fat diet to dissect epididymal versus testicular contributions to the sperm sncRNA pool and offspring health. We show that epididymal spermatozoa, but not developing germ cells, are sensitive to the environment and identify mitochondrial tRNAs (mt-tRNAs) and their fragments (mt-tsRNAs) as sperm-borne factors. In humans, mt-tsRNAs in spermatozoa correlate with body mass index, and paternal overweight at conception doubles offspring obesity risk and compromises metabolic health. Sperm sncRNA sequencing of mice mutant for genes involved in mitochondrial function, and metabolic phenotyping of their wild-type offspring, suggest that the upregulation of mt-tsRNAs is downstream of mitochondrial dysfunction. Single-embryo transcriptomics of genetically hybrid two-cell embryos demonstrated sperm-to-oocyte transfer of mt-tRNAs at fertilization and suggested their involvement in the control of early-embryo transcription. Our study supports the importance of paternal health at conception for offspring metabolism, shows that mt-tRNAs are diet-induced and sperm-borne and demonstrates, in a physiological setting, father-to-offspring transfer of sperm mitochondrial RNAs at fertilization.

The design of new tokamak machines relying on the use of high temperature superconductors (HTS) is promoting the study of HTS properties at the operating conditions required by fusion applications. In particular, the interest in the damage induced by neutron irradiation on REBa2Cu3O7-δ (REBCO, RE = Y or lanthanide series), one of the most used family of HTS, has recently risen and several studies have been devoted to radiation hardness tests performed with ion irradiation or fission neutrons. In this work, the effect of neutron irradiation on YBCO films and commercial REBCO tapes was investigated using, for the first time, neutrons produced by the D-T fusion reaction. The experiment was carried out at ENEA-Frascati Neutron Generator (FNG) where a deuteron beam is accelerated up to 300 keV and directed on a tritiated target to produce a nearly isotropic 14.1 MeV neutron field via the T(d,n)α fusion reaction. Different YBCO films deposited through metal-organic decomposition (MOD) route on single crystals (SrTiO3 and LaAlO3) and REBCO commercial tapes, grown by pulsed laser deposition, were irradiated. Samples exposed to three fluences were compared with a maximum neutron fluence of 1.2·1014 cm−2. The properties of HTS materials were assessed before and after irradiation by means of different techniques. From these measurements, no significant effect on the considered properties was recognized indicating the robustness of films up to the explored irradiation fluences.

•Clinical characteristics and fosfomycin synergism of KPC-variants were investigated.•KPC-variant detection follows a previous CZA-susceptible KPC-Kp in the same patient.•Treatment with meropenem achieved clinical cure in all the patients.•Fosfomycin plus meropenem resulted in full synergism in 40% of cases.•FOS plus MEM may be an option for KPC-variant infections at low risk of mortality.

Purpose: Salivary gland (SG) tissue and derived neoplasms may occur in the sellar region. As the current literature is mostly limited to case reports, the puzzling case of an inflammatory SG removed by transsphenoidal surgery (TS) and mimicking a prolactinoma prompted us to perform the first systematic review of these unusual conditions. Methods: A systematic literature search was conducted according to the PRISMA guidelines. Forty-four individual cases—non-neoplastic enlarged salivary glands (NNESG, n= 15), primary benign (n= 7) and malignant (n= 8) ectopic salivary tumours (ST) and sellar metastasis from eutopic primary ST (n= 14)—were suitable for the analysis of clinical, radiological and pathological characteristics. Therapeutic outcome was reviewed as a secondary endpoint. Results: All cases were diagnosed after surgery. NNESG commonly affected young and/or female patients, typically leading to headaches and hyperprolactinemia and originating close to the neurohypophysis. Submucosal SG should be excluded before concluding to an intrasellar NNESG after TS. No gender or age predominance was found for primary ectopic ST, which present as large tumors, with histological phenotypes similar to common ST. Hypopituitarism and diabetes insipidus were more frequent in ST than in NNESG. NNESG and benign ectopic ST rarely recur. Malignant ectopic ST should be distinguished from secondary localizations of eutopic ST reaching the sella by contiguity or metastatic spread; both share a frequent unfavorable outcome. Conclusion: Sellar neoplasms derived from SG are rare but misleading conditions and pituitary dysfunction is likely to be more common than currently reported. Appropriate pathological evaluation and multidisciplinary approach are required.

This study examines the expression of three microRNAs (hsa-miR-661, hsa-miR-21-5p, hsa-miR-372-5p) in spent pre-implantation embryos culture media to identify possible new non-invasive biomarkers of embryo competence, predictive of development to the blastocyst stage. A preliminary analysis on 16 patients undergoing IVF cycles was performed by collecting and stored spent culture media on the fifth/sixth day of embryo culture. Expression of miRNAs was evaluated according to the embryos’ fate: 1) NE/DG: non-evolved or degenerate embryos; 2) BLOK: embryos developed to the blastocyst stage. Preliminary results revealed a higher miRNAs expression in NE/DG spent media. To elucidate the roles of these miRNAs, we employed a robust bioinformatics pipeline involving: 1) in-silico miRNA Target Prediction using RNAHybrid, which identified the most-likely gene targets; 2) Construction of a Protein-Protein Interaction network via GeneMania, linking genes with significant biological correlations; 3) application of modularity-based clustering with the gLay app in Cytoscape, resulting in three size-adapted subnets for focused analysis; 4) Enrichment Analysis to discern the biological pathways influenced by the miRNAs. Our bioinformatics analysis revealed that hsa-miR-661 was closely associated with pathways regulating cell shape and morphogenesis of the epithelial sheet. These data suggest the potential use of certain miRNAs to identify embryos with a higher likelihood of developing to the blastocyst stage. Further analysis will be necessary to explore the reproducibility of these findings and to understand if miRNAs here investigated can be used as biomarkers for embryo selection before implantation into the uterus or if they may be reliable predictors of IVF outcome.

Dietary intake of methyl donors, such as folic acid and methionine, shows considerable intra-individual variation in human populations. While it is recognized that maternal departures from the optimum of dietary methyl donor intake can increase the risk for mental health issues and neurological disorders in offspring, it has not been explored whether paternal dietary methyl donor intake influences behavioral and cognitive functions in the next generation. Here, we report that elevated paternal dietary methyl donor intake in a mouse model, transiently applied prior to mating, resulted in offspring animals (methyl donor-rich diet (MD) F1 mice) with deficits in hippocampus-dependent learning and memory, impaired hippocampal synaptic plasticity and reduced hippocampal theta oscillations. Gene expression analyses revealed altered expression of the methionine adenosyltransferase Mat2a and BK channel subunit Kcnmb2, which was associated with changes in Kcnmb2 promoter methylation in MD F1 mice. Hippocampal overexpression of Kcnmb2 in MD F1 mice ameliorated altered spatial learning and memory, supporting a role of this BK channel subunit in the MD F1 behavioral phenotype. Behavioral and gene expression changes did not extend into the F2 offspring generation. Together, our data indicate that paternal dietary factors influence cognitive and neural functions in the offspring generation.

Background/Objectives:Dieting is a popular yet often ineffective way to lower body weight, as the majority of people regain most of their pre-dieting weights in a relatively short time. The underlying molecular mechanisms driving weight regain and the increased risk for metabolic disease are still incompletely understood. Here we investigate the molecular alterations inherited from a history of obesity.Methods:In our model, male high-fat diet (HFD)-fed obese C57BL/6J mice were switched to a low caloric chow diet, resulting in a decline of body weight to that of lean mice. We measured body composition, as well as metrics of glucose, insulin and lipid homeostasis. This was accompanied by histological and gene expression analysis of adipose tissue and liver to assess adipose tissue inflammation and hepatosteatosis. Moreover, acute hypothalamic response to (re-) exposure to HFD was assessed by qPCR.Results & Conclusions:Within 7 weeks after diet switch, most obesity-associated phenotypes, such as body mass, glucose intolerance and blood metabolite levels were reversed. However, hepatic inflammation, hepatic steatosis as well as hypertrophy and inflammation of perigonadal, but not subcutaneous, adipocytes persisted in formerly obese mice. Transcriptional profiling of liver and perigonadal fat revealed an upregulation of pathways associated with immune function and cellularity. Thus, we show that weight reduction leaves signs of inflammation in liver and perigonadal fat, indicating that persisting proinflammatory signals in liver and adipose tissue could contribute to an increased risk of formerly obese subjects to develop the metabolic syndrome upon recurring weight gain.

Obplacental giant cells are enlarged cells, found following implantation, in the antimesometrial region of the rabbit uterus. They probably originate from trophoblastic knobs that traverse the uterine epithelium during early implantation. Little is known about their function. In this study, trophoblast, placental, paraplacental and obplacental tissues at days 7–15 post-coitum, and enzyme-isolated giant cells at day 15 were studied by two-dimensional gel electrophoresis, followed by immunoblotting and light-microscopic immunohistochemistry, for the presence of human chorionic gonado- tropin-like proteins. Immunostaining was performed by using anti-human chorionic gonadotropin antibodies. In gel electrophoresis of obplacental tissue and isolated giant cells, two proteins of human chorionic gonadotropin-like antigenicity at 26?kDa with pIs equivalent to pH?6.4 and 6.6 were found; they were absent in the placenta, paraplacenta, day-7 blastocyst and day-8 trophoblast. The onset of synthesis of these proteins could be observed when day-8 trophoblastic tissue was cultured in vitro for 24?h. In immunohistochemistry, only the obplacental giant cells showed a positive reaction, indicating that the production of chorionic gonadotropin occurs in this cell type.

The aim of this study is to show and evaluate the combined procedure, which uses an endoscopic suprapubic access and a surgical perineal access to repair posterior urethral stricture secondary to traumatic pelvis fracture.Material and Methods In the period from January 1989 to December 2007 eighty-nine patients underwent urethral surgery for post-traumatic posterior urethral stricture. From January 2003 all patients underwent combined endoscopic and surgical technique. According to this technique, the patient is placed in simple lithotomic position with the calves carefully placed in Allen stirrups. Two surgical teams work simultaneously. A middle-line perineal incision is made and the bulbar urethra is isolated proximally and the membranous urethra is transected at the strictured site. At the same time the second surgical team performs an endoscopic suprapubic access placing the “Amplatz” sheath, previous progressive dilation to 20/22 Ch. By using a rigid or flexible cystoscope the operator follows endoscopically the bladder neck and reaches the stenotic site performing an anterograde urethroscopy. At this point the perineum is transilluminated by the endoscope and the surgeon can easily identify the proximal urethral end. A soft guide wire is inserted at this point into the urethra through the endoscope to facilitate the dilation till a nose speculum can be inserted. At this point an end-to-end anastomosis is performed. A Foley 18 Fr catheter and a suprapubic cystostomy are left in place for 1 month; a voiding cystourethrography is then performed.Results The bulboprostatic anastomosis shows better results (65% of success) if compared with the other techniques (Badenoch, two stage urethroplasty, perineal urethrostomy). A definite increase in the success rate (10%) has been evident in the last five years, simultaneously to the use of combined technique.Conclusions The combined perineal and suprapubic access, in post-traumatic posterior urethral strictures repair, allows achieving a better and easy location and a better preparation of the proximal urethra. The final target is to obtain a better bulboprostatic anastomosis, with better results confirmed by long-term follow-up. In particular, the endoscopic management of the suprapubic access is possible and of minor invasiveness to the patient.

The objective of the present study is to test the use of a haemostatic sponge for urethral reconstructive surgery and to assess the relevant benefits.Methods. The haemostatic sponge is made up of collagen with human fibrinogen and human thrombin. When the sponge gets in contact with fluids, such as blood, lymph or saline solution, the fibrinogen and thrombin are activated and form a fibrin net able to achieve local haemostasis and tissue regeneration. This kind of product was used in 30 selected patients (pts.), aged 19 - 70 (mean age: 35), who underwent single-stage anastomotic urethroplasty from January 2006 to March 2007: 22 bulbar end-to-end anastomoses and 8 bulbo-prostatic anastomoses. All operations were performed due to post-traumatic stricture of bulbar and posterior urethra. The main selection criterion for the use of the haemostatic sponge has always been the tendency to and/or the presence of bleeding in the different surgical stages. The sponge imbibed in saline solution and suitably modeled for all specific requirements was differently placed: sleeve-shaped in the case of bulbar anastomoses (16 pts.); as a “patch” in the centre of spongioplasty in bulbo-prostatic anastomoses (8 pts.), and within the widening incisions of the urethral roof performed between the two cavernous bodies at level of the intercrural septum (5 pts.). In 1 case (obese patient) a single sponge was placed after the deep perineal reconstruction on the bulbo-cavernous muscle-subcutaneous layers because of massive bleeding.Results. In all cases a perfect control of haemostasis could be achieved, with immediate end of bleeding in the points where the haemostatic sponges were used. The dressings performed on day 3 upon removal of the compressive scrotum-perineal bandages did not show any late post-operative bleeding. The follow-up visits at month 1 and 3 yielded positive outcomes for all 30 patients, who achieved complete wound healing by first intention, and a perfect anastomosis especially in the bulbar end-to-end urethroplasty, confirmed by urethrografy at one month.Conclusions. The association of human fibrinogen and human thrombin in haemostatic sponges represents a manageable, useful product, apparently sure, not negatively interfering in repairing and regenerating tissue processes, and allowing a simple and direct control of important bleeding events occurring during urethral reconstructive surgery as well as other operations. It is therefore advisable to more widely use the product after a suitable follow-up period.

Formation and inactivation of testosterone is performed by various members of the 17β-hydroxysteroid dehydrogenase (17β-HSD) family. The main player in testosterone formation is considered to be 17β-HSD type 3, which catalyzes the reduction of androstenedione to testosterone with high efficiency and is almost exclusively expressed in testis. So far, only the mammalian homologs have been characterized but nothing is known about the role of 17β-HSD type 3 in other vertebrates. In this study, we describe the identification and characterization of the zebrafish homolog. We found zebrafish 17β-HSD type 3 to be expressed in embryogenesis from sphere to 84 h post-fertilization. Expression was also detected in various tissues of both male and female adults, but displayed sexual dimorphism. Interestingly, expression was not highest in male testis but in male liver. In female adults, strongest expression was observed in ovaries. At the subcellular level, both human and zebrafish 17β-HSD type 3 localize to the endoplasmic reticulum. The zebrafish enzyme in vitroeffectively catalyzed the conversion of androstenedione to testosterone by use of NADPH as cofactor. Among further tested androgens epiandrosterone and dehydroepiandrosterone were accepted as substrates and reduced at C-17 by the human and the zebrafish enzyme. Androsterone and androstanedione though, were only substrates of human 17β-HSD type 3, not the zebrafish enzyme. Furthermore, we found that both enzymes can reduce 11-ketoandrostenedione as well as 11β-hydroxyandrostenedione at C-17 to the respective testosterone forms. Our results suggest that 17β-HSD type 3 might play slightly different roles in zebrafish compared with human although testosterone itself is likely to have similar functions in both organisms.

The therapeutic potential of bone marrow–derived stromal cells for the therapy of Tay-Sachs disease is primarily related to the restoration of their own GM2 ganglioside storage. With this aim, we produced bone marrow–derived stromal cells from the adult Tay-Sachs animal model and transduced them with a retroviral vector encoding for the α-subunit of the lysosomal enzyme β-hexosaminidase A (E.C. 3.2.1.52). Our results demonstrate that transduced Tay-Sachs bone marrow–derived stromal cells have β-hexosaminidase A comparable to that of bone marrow-derived stromal cells from wild-type mice. Moreover, β-hexosaminidase A in transduced Tay-Sachs bone marrow-derived stromal cells was able to hydrolyze the GM2 ganglioside in a feeding experiment, thus demonstrating the correction of the altered phenotype.

Microarray-based techniques allow us to visualize and quantify the expression of every single gene in any population of cells. In yeast the true potential of large-scale transcriptome analysis in identifying regulatory units and understanding gene function has already been demonstrated by evaluating expression profiles of a comprehensive group of mutants. We discuss the potential of DNA-chip technologies for the analysis of gene expression in complex organisms. The usefulness of transcriptome analysis for clinical purposes und diagnosis of cancers is already well established. We argue that microarray-based expression profiling will also be a useful tool for the analysis of gene function and approaches complementary to classical phenotypic description in mammals, particularly in regard of the large resources of mutant models that are currently being generated by gene-targeting and mutagenesis of the mouse genome. Experimental requirements and potential future directions are discussed.

Abstract:: We have developed and characterized a new method to produce a continuous beam of cold atoms from a standard vapour-cell magneto-optical trap (MOT). The experimental apparatus is very simple. Using a single laser beam it is possible to hollow out in the source MOT a direction of unbalanced radiation pressure along which cold atoms can be accelerated out of the trap. The transverse cooling process that takes place during the extraction reduces the beam divergence. The atomic beam is used to load a magneto-optical trap operating in an ultra-high vacuum environment. At a vapour pressure of 10-8mbar in the loading cell, we have produced a continuous flux of 7×107atoms/s at the recapture cell with a mean velocity of 14 m/s. A comparison of this method with a pulsed transfer scheme is presented.

Abstract:: We report a new method to generate an optical dipole potential with a null intensity region surrounded in all directions by light walls. This is achieved with a simple scheme based on a conical lens. Applications to optical trapping of neutral atoms are discussed.

Canestrato Pugliese cheeses from ewe milk were produced according to a traditional protocol and by adding 7.0 log10cfu of fresh cells per gram of Bifidobacterium bifidumBb02, Bifidobacterium longumBb46, or both species. The traditional technology was modified slightly to favor the survival of probiotic microorganisms. After 56 d of ripening, the survival of B. bifidumBb02 and B. longumBb46 was 6.0 and 5.0 log10cfu/g, respectively. After 19 d cheeses contained ca. 7.0 log10cfu/g of bifidobacteria. Compared to traditional cheese, the addition of bifidobacteria seemed to support the growth and survival of mesophilic lactobacilli and Streptococcus thermophilus, used as starter, during ripening. No significant differences were observed in the main chemical composition, and only a slightly higher concentration of acetic acid was found in cheeses with bifidobacteria added. On the contrary, α- and &szligbeta;-galactosidase activities were markedly more pronounced in the presence of bifidobacteria, especially with B. bifidumBb02. In contrast with traditional cheese, the lactose was completely hydrolyzed in cheeses made with bifidobacteria. Urea-PAGE electrophoresis of the pH 4.6-soluble and pH 4.6-insoluble N fractions did not show appreciable variations. Only the reversed-phase-HPLC analysis of the pH 4.6-soluble N showed a slightly more complex profile in the presence of bifido-bacteria. This finding was in agreement with the higher value of the pH 4.6-soluble N/total N ratio and with the more pronounced amino-, imino-, and dipeptidase activities found in all the cheeses with the bifidobacteria added, especially B. bifidumBb02. No differences were found in the free amino acid and free fatty acid contents. The amino acids glutamic acid, valine, proline, leucine, and lysine and the fatty acids butyric, caproic, capric, and oleic acids were found at the highest concentrations. The sensory evaluation did not show significant differences, and Canestrato Pugliese cheeses were characterized by small and uniformly distributed eyes, were pale yellow, had an elastic consistency and a Pecorino-like smell, were very salty, andtended to be moderately piquant.

One hundred and seventy strains of sourdough lactobacilli were screened for their capacity to grow optimally by using pentoses. Lb. alimentariusM106 and M137, Lb. hilgardiiS32, Lb. brevisAM11, Lb. fermentumI4 and Lb. brevisAM8 had a higher cell yield, growth rate and acetic acid production on a mix of arabinose, xylose or ribose and maltose than on maltose as carbon sources. In the cofermentation process, pentoses were preferentially consumed instead of maltose. Pentosan extract was treated with α-L-arabinofuranosidase from Aspergillus nigeror endo-xylanase from Bacillus subtilisto produce hydrolysates respectively each of arabinose and xylose. Depending on the type of pentose and other unidentified compounds, hydrolysates specifically substantiated the growth and acetic acid production of sourdough lactobacilli. Sourdough fermentation by Lb. hilgardiiS32 with addition of pentosans and endo-xylanase enzyme increased the acidification rate, titratable acidity, acetic acid content and cell yield number. In addition to the well known role in retarding bread staling, pentosans may also be used as sources of pentoses which favour the performances of selected sourdough lactobacilli.

Abstract:  Autolysis of Lactobacillus sanfranciscensis in phosphate buffer was strain-dependent and maximal in the exponential growth phase and at pH 5.6. It decreased to 15% of the maximum at pH 4.5. In culture medium, autolysis did not correspond to the cell death rate because the low pH reached after fermentation limited it markedly. Appreciable autolysis was only found when buffering citrate or fumarate were added to the culture medium. Leakage of peptidases from cells to the culture medium confirmed the autolysis. Renaturating SDS-PAGE showed an autolytic profile composed of four clean lytic bands of ca. 102, 87, 76.5 and 61.5 kDa. This profile was observed in all strains and did not vary with the growth phase or with the use of the crude cell-envelope or crude extracts. Sourdough fermentations lasting 24–48 h showed no differences in the leakage of peptidases when strains which differed greatly for autolysis were compared. However, if citrate was added to the dough it enhanced the release of intracellular peptidases by fast autolytic strains because of the less acidic conditions which favored microbial autolysis during sourdough fermentation.

Abstract: The latency phase, growth rate, cell yield and end-products of Lactobacillus sanfranciscensis CB1 were affected by oxygen and the supply of 225 μM Mn2+. Mn2+ was especially related to the highest substrate consumption. Aerobiosis and Mn2+ supply were responsible for the highest superoxide dismutase activity. An auto-inhibitory accumulation of H2O2 meant that the survival of air-grown cells supplied with Mn2+ rapidly decreased during the stationary phase. As shown by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, Mn2+ supply influenced protein expression. As shown by non-denaturating zymograms, Lb. sanfranciscensis CB1 expressed an approximately 12.5-kDa superoxide dismutase, which is probably Mn-dependent. The enzyme was insensitive to H2O2 treatment, was not induced by the presence of paraquat under aerobic conditions and was relatively stable at pH 4.0. Sourdoughs that contained high levels of oxygen enhanced cell growth, acidification and acetic acid production by Lb. sanfranciscensis CB1.

Creation of new human faces for synthetic actors is a tedious and painful task. The situation may be improved by introducing tools for the creation. Two approaches are discussed in this paper: modification and edition of an existing synthetic actor using local transformations; generation of new synthetic actors obtained by interpolation between two existing actors; creation of a synthetic actor by composition of different parts. This paper also describes the methods used in the facial animation of synthetic actors who change their personalities from one person to another. This means that our purpose is to transform one character into another, and also to transform the animation at the same time. The interpolation must be at several levels: the shape level, the parameter level, the expression level and the script level. For the animation, we introduce three levels of inbetweens: inbetween parameters, inbetween expressions and inbetween scripts. The method has been completely implemented and integrated into the Human Factory software.

Culturing of rabbit pre-implantation embryos was performed in Ham's F10 medium supplemented with polyvinylpyrrolidone. Under these culture conditions, day 6 post coitum blastocysts increased their diameter within 24 h to 80% of that of day 7 blastocysts grown in vivo. Despite this substained growth, the embryonic disc remained undifferentiated with clear signs of degeneration after 24 h of culture. Basic fibroblast growth factor (bFGF) was able to overcome this developmental block. After 12 h of culture, day 6 blastocysts showed pear-shaped embryonic discs, and after 24 h, the primitive streak with Hensen's node was visible. The bFGF had no comparable effects on day 5 and day 7 blastocysts. The embryonic discs of day 5 blastocysts degenerated, even in the presence of bFGF, whereas day 7 blastocysts were able to form their primitive streak, also in the absence of bFGF. TGFß1 did not promote embryonic development in vitro. The data indicate that the onset of mesoderm formation in the rabbit is controlled by a growth factor of the FGF-family.

Bifidobacterium bifidum, Bifidobacterium infantis, and Bifidobacterium longumwere incorporated into Crescenza cheese, individually or as multispecies mixtures and as free cells or as cells immobilized in calcium alginate gel. The viability of the bifidobacteria differed in 14-d-old cheeses: when added individually, the cell counts of B. bifidum, B. longum, and B. infantiswere log108.05, 7.12, and 5.23 cfu/g, respectively. The multispecies mixtures only reached about log105.0 cfu/g of cheese. The presence of bifidobacteria did not influence the aerobic microflora, the growth of Streptococcus thermophilusused as starter, or the gross composition of the cheese. No differences were found in the primary proteolysis with respect to the conventional production of Crescenza cheese, but higher levels of pH 4.6-soluble N and more pronounced activities of aminopeptidase, iminopeptidase, dipeptidase, and tripeptidase were detected in all of the cheeses with added bifidobacteria. Crescenza cheeses with added bifidobacteria contained only traces of lactose and had slightly higher concentrations of lactic and acetic acids than did the conventional cheese. The presence of bifidobacteria was also related to high α- and β-galactosidase activities in the cheese. Sensory evaluation of the cheeses with incorporated bifidobacteria was very similar to that of Crescenza cheese produced by the conventional method.

The Drosophila Delta (Dl) gene is essential for cell-cell communication regulating the determination of various cell fates during development. Dl encodes a transmembrane protein, which contains tandem arrays of epidermal-growth-factor-like repeats in the extracellular domain and directly interacts with Notch, another transmembrane protein with similar structural features, in a ligand-receptor-like manner. Similarly, cell-cell interactions involving Delta-like and Notch-like proteins are required for cell fate determinations in C. elegans. Notch homologues were also isolated from several vertebrate species, suggesting that cell-to-cell signaling mediated by Delta- and Notch-like proteins could also underlie cell fate determination during vertebrate development. However, in vertebrates, no Delta homologues have yet been described. We have isolated a novel mouse gene, Dll1 (delta-like gene 1), which maps to the mouse t-complex and whose deduced amino acid sequence strongly suggests that Dll1 represents a mammalian gene closely related to Drosophila Delta. Dll1 is transiently expressed during gastrulation and early organogenesis, and in a tissue-restricted manner in adult animals. Between day 7 and 12.5 of development, expression was detected in the paraxial mesoderm, closely correlated with somitogenesis, and in subsets of cells in the nervous system. In adult animals, transcripts were detected in lung and heart. Dll1 expression in the paraxial mesoderm and nervous system is strikingly similar to the expression of mouse Notch1 during gastrulation and early organogenesis. The overlapping expression patterns of the Dll1 and Notch1 genes suggest that cells in these tissues can communicate by interaction of the Dll1 and Notch1 proteins. Our results support the idea that Delta- and Notch-like proteins are involved in cell-to-cell communication in mammalian embryos and suggest a role for these proteins in cellular interactions underlying somitogenesis and development of the nervous system.

Continuously tunable ultraviolet laser radiation at 397 nm was generated by doubling the output of a semiconductor diode laser. The fundamental radiation was provided by a 150 mW AlGaAs laser diode injected by a low-power AlGaAs laser diode which was frequency stabilized by optical feedback using a new scheme of a miniature external cavity. Second-harmonic generation was produced in a lithium-triborate crystal placed in a compact enhancement cavity. The fundamental radiation was used for sub-Doppler spectroscopy of the Ar I 4s3P00-4p1P1 transition at 795 nm; the second-harmonic radiation was used for spectroscopy of the Ca II 42S1/2-42P1/2 transition at 397 nm.

Embryonic and fetal skeletal myoblasts were grown in culture in the presence of TGF beta. Under the conditions employed, TGF beta inhibited differentiation of fetal but not of embryonic myoblasts. To investigate the possible relevance of these data to skeletal muscle histogenesis in vivo, we studied the proliferation/differentiation state of mesodermal cells in the proximal region of the limb bud at the time of primary fiber formation. BrdU labeling and immunostaining for myosin heavy chains revealed that very few mesodermal cells enter the S phase of the cycle when differentiated primary fibers first appear. However, a few hours later, many cells in S phase surround newly formed muscle fibers, suggesting that the latter may be a source of mitogens for undifferentiated myoblasts. Co-culture experiments supported this hypothesis, showing that medium conditioned by fiber-containing explants can stimulate myoblast proliferation. Taken together these data suggested a possible mechanism for the regulation of muscle fiber formation. The model assumes that fibers form in the proximal region of the limb bud, where TGF beta is known to be present, and BrdU labeling experiments did not reveal cells in S phase. It is conceivable that non-dividing embryonic myoblasts (which do not respond to TGF beta) can undergo differentiation, while fetal myoblasts are inhibited by TGF beta. Once formed, primary fibers may stimulate a new wave of proliferation in fetal myoblasts, in order to expand the pool of cells needed to form secondary fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Rabbit blastocysts of day 6 post coitum were cultured in a chemically defined, protein-free medium for 24 h. Although the trophoblast continued to grow, the embryonic disc degenerated. Addition of basic fibroblast growth factor (FGF-2, of human recombinant or bovine origin, 10 ng/ml) to the culture medium resulted in significant developmental progress. The embryonic disc became pear-shaped showing a round anterior edge and a posterior node. The primitive streak and Hensen’s node indicated that gastrulation had begun. Mesoderm formation was confirmed from histological sections and by localization of the expression of T-gene transcripts in whole-mount preparations. FGF-2 mRNA was detected in both day-6 endometrium and day 6-blastocysts using in vitro translation followed by immunoprecipitation with a monoclonal antibody to FGF-2. In the uterine secretions of day-6 pregnant and pseudopregnant animals, several proteins exhibiting FGF-2 antigenicity were detected on Western blots following two-dimensional gel electrophoresis. As day-6 blastocysts required exogenous FGF-2 in vitro and as FGF-2 of uterine origin is present in the uterine secretion, the maternal growth factor can promote gastrulation in vivo.

A recent study has shown the reproducibility of time to exhaustion (time limit: ttime) at the lowest velocity that elicits the maximal oxygen consumption (υv˙O2 max). The same study found an inverse relationship between this time to exhaustion at υv˙O2 max and υv˙O2 max among 38 élite long-distance runners (Billat et al. 1994b). The purpose of the present study was to compare the time to exhaustion at the power output (or velocity) at v˙O2 max for different values of v˙O2 max, depending on the type of exercise and not only on the aerobic capacity. The time of exhaustion at υv˙O2 max (tlim) has been measured among 41 élite (national level) sportsmen: 9 cyclists, 9 kayak paddlers, 9 swimmers and 14 runners using specific ergometers. Velocity or power at v˙O2 max ( υv˙O2 max) was determined by continuous incremental testing. This protocol had steps of 2 min and increments of 50 W, 30 W, 0.05ms- and 2km- for cyclists, kayak paddlers, swimmers and runners, respectively. One week later, dim was determined under the same conditions. After a warm-up of 10 min at 60% of their υv˙O2 max, subjects were concluded (in less than 45 s) to their υv˙O2 max and then had to sustain it as long as possible until exhaustion. Mean values of υv˙O2 max and dim were respectively equal to 419±49 W (tlim = 222 ± 91 s), 239±56W ( tlim = 376 ± 134 s), l 46±009ms-1 ( tlim = 287± 160s) and 22.4 ±0.8kmh-1 ( tlim = 321 ±84s), for cyclists, kayak paddlers, swimmers and runners. Time to exhaustion at υv˙O2 max was only significantly different between cycling and kayaking (ANOVA test, p<0.05). Otherwise, υv˙O2max (expressed in ml min-1 kg-1) was significantly different between all sports except between cycling and running (p < 0.05). In this study, time to exhaustion at υv˙O2 max was also inversely related to υv˙O2 max for die entire group of elite sportsmen (r= -0.320, p<0.05, n = 41). The inverse relationship between υv˙O2 max and dim at υv˙O2 max has to be explained, it seems that dim depends on υv˙O2 max regardless of the type of exercise undertaken.

The accumulation of two myogenic regulatory proteins, MyoD and myogenin, was investigated by double-immunocytochemistry and correlated with myosin heavy chain expression in different classes of myoblasts in culture and during early myogenesis in vivo. During in vitro differentiation of fetal myoblasts, MyoD-positive cells were detected first, followed by the appearance of cells positive for both MyoD and myogenin and finally by the appearance of differentiated myocytes and myotubes expressing myosin heavy chain (MHC). A similar pattern of expression was observed in cultures of embryonic and satellite cells. In contrast, most myogenic cells isolated from newly formed somites, expressed MHC in the absence of detectable levels of myogenin or MyoD. In vivo, the appearance of both myogenin and MyoD proteins was only detected at 10.5 d postcoitum (d.p.c.), when terminally differentiated muscle cells could already be identified in the myotome. Parasagittal sections of the caudal myotomes of 10.5-d-old embryos showed that expression of contractile proteins preceded the expression of myogenin or MyoD and, when coexpressed, MHC and myogenin did not co-localize within all the cells of the myotome. In the limb bud, however, many myogenin (or MyoD) positive/MHC negative cells could be observed in the proximal region at day 11. During further embryonic development the expression of these proteins remained constant in all the muscle anlagens examined, decreasing to a low level during the late fetal period. Western and Northern analysis confirmed that the myogenin protein could only be detected after 10.5 d.p.c. while the corresponding message was clearly present at 9.5 d.p.c., strongly suggesting a posttranscriptional regulation of myogenin during this stage of embryonic development. These data show that the first myogenic cells which appear in the mouse myotome, and can be cultured from it, accumulate muscle structural proteins in their cytoplasm without expressing detectable levels of myogenin protein (although the message is clearly accumulated). Neither MyoD message or protein are detectable in these cells, which may represent a distinct myogenic population whose role in development remains to be established.

•In real life, DMF reduced the annualized relapse rate (ARR) by 75% respect to the pre-treatment ARR.•Age of onset of MS was related with rate or relapses, with younger age being protective.•De-escalating from second-line therapies was associated to higher risk of relapsing.•Disease duration was associated with higher rate of NEDA-3 failure.•Higher pre-treatment EDSS was associated with DMF discontinuation.

We demonstrate accurate two-dimensional mapping of the phase retardation induced by the electro-optic effect in lithium niobate crystals. Off-axis digital holography is used to investigate congruent z-cut crystals. The spatially resolved optical path difference is interferometrically measured while a linearly rising voltage ramp is applied to the crystal. This procedure provides information on the uniformity of crystals’ electro-optic properties and offers the ability to detect the presence of defects that is of fundamental importance for reliable processing of photonic devices.

Explosive volcanic eruptions can follow long cycles of activity1, and the material that they inject into the atmosphere may affect the climate2. We report here the discovery of ultrathin micrometre-sized glass shards from such eruptions, extracted from both ancient Antarctic ices and recent snow samples from Greenland, in which they have been well preserved by ‘deep freezing’. When such shards have been preselected using a high-voltage electron microscope, microanalysis of their eight major constituent elements gives new clues about the origin of major volcanic acid fallout previously detected in the cores (including important characteristics of their parent eruptions), and reveals complex volcanic ash layers that could reflect periods of world-wide enhanced volcanicity. Moreover, the very small Reynolds numbers of the micrometre-sized shards make them promising tracers of dust transport processes in the ancient atmosphere.

The poem "A Settling Walk," by M. J. De Angelis is presented. First Line: When I needed all things to make sense; Last Line: Though, I'm waiting for things to make sense.