Sesquiterpene lactones and other constituents from Schistostephium species

From four Schistostephium species, in addition to known compounds, 43 new ones were isolated, three isocomene, two bisabolene, two eudesmane, two allo-himachalene and two nerolidol derivatives, two sesquiterpenes with a new carbon skeleton derived from isocomene, nine eudesmanolides, 20 6,12- and 8,12-germacranolides respectively, and a prostaglandin-like acid. The structures were elucidated by spectroscopic methods, especially high field ¹H NMR and some chemical transformations. The stereochemistry of some 8,12-germacranolides was established by partial synthesis which required revision of the previous assignments of the configuration of inunolide, its 4,5- and 1,10-epoxide, vernudifloride, simsiolide, 6-hydroxy-1,10-epoxyinunolide and perhaps also of tanachin. The chemotaxonomic situation is discussed briefly.     

Birth of African Wildcat cloned kittens born from domestic cats

In the present study, we used the African Wildcat (Felis silvestris lybica) as a somatic cell donor to evaluate the in vivo developmental competence, after transfer into domestic cat recipients, of cloned embryos produced by the fusion of African Wildcat (AWC) fibroblast cell nuclei with domestic cat cytoplasts. Cloned embryos were produced by fusion of a single AWC somatic cell to in vivo or in vitro enucleated domestic cat cytoplasts. When the two sources of oocytes were compared, fusion rate was higher using in vivo-matured oocytes as recipient cytoplasts, but cleavage rate was higher after reconstruction of in vitro-matured oocytes. To determine the number of reconstructed embryos required per domestic cat recipient to consistently establish pregnancies, AWC cloned embryos were transferred within two groups: recipients (n = 24) receiving < or =25 embryos and recipients (n = 26) receiving > or =30 embryos. Twelve recipients (46.2%) receiving > or =30 embryos were diagnosed to be pregnant, while no pregnancies were established in recipients receiving < or =25 NT embryos. Also, to determine the influence of length of in vitro culture on pregnancy rate, we compared oviductal transfer on day 1 and uterine transfer on day 5, 6, or 7. Pregnancy rates were similar after transfer of embryos on day 1 (6/12; 50.0%), day 5 (4/9; 44.4%), or day 6 (2/5; 40.0%) to synchronous recipients, but the number of fetuses developing after transfer of embryos on day 1 (n = 17), versus day 5 (n = 4) or day 6 (n = 3) was significantly different. Of the 12 pregnant recipients, nine (75%) developed to term and fetal resorption or abortion occurred in the other three (25%) from day 30 to 48 of gestation. Of a total of 17 cloned kittens born, seven were stillborn, eight died within hours of delivery or up to 6 weeks of age, and two are alive and healthy. Perinatal mortality was due to lung immaturity at premature delivery, placental separation and bacterial septicemia. Subsequent DNA analysis of 12 cat-specific microsatellite loci confirmed that all 17 kittens were clones of the AWC donor male. These AWC kittens represent the first wild carnivores to be produced by nuclear transfer.     

Glass bead purification of plasmid template DNA for high throughput sequencing of mammalian genomes

To meet the new challenge of generating the draft sequences of mammalian genomes, we describe the development of a novel high throughput 96-well method for the purification of plasmid DNA template using size-fractionated, acid-washed glass beads. Unlike most previously described approaches, the current method has been designed and optimized to facilitate the direct binding of alcohol-precipitated plasmid DNA to glass beads from alkaline lysed bacterial cells containing the insoluble cellular aggregate material. Eliminating the tedious step of separating the cleared lysate significantly simplifies the method and improves throughput and reliability. During a 4 month period of 96-capillary DNA sequencing of the Rattus norvegicus genome at the Baylor College of Medicine Human Genome Sequencing Center, the average success rate and read length derived from >1 800 000 plasmid DNA templates prepared by the direct lysis/glass bead method were 82.2% and 516 bases, respectively. The cost of this direct lysis/glass bead method in September 2001 was ~10 cents per clone, which is a significant cost saving in high throughput genomic sequencing efforts.     

Maternal Chlamydia trachomatis Infections and Preterm Births in a University Hospital in Vitoria,Brazil

Background

Preterm birth (PTB) is a major determinant of neonatal morbimortality with adverse consequences for health. The causes are multifactorial, with intrauterine infection probably explaining most of these outcomes. It is believed that infection with Chlamydia trachomatis (CT) is also involved in PTB and premature rupture of membranes.

Objetives

To evaluate the prevalence of and associated factors for CT among cases of PTB attended at a University Hospital in Vitoria, Brazil.

Methods

A cross-sectional study performed among parturient who had preterm birth from June 2012 to August 2013 in Vitoria, Brazil. Participants answered a questionnaire including demographic, behavioral, and clinical data. A sample of urine was collected and screened for CT using polymerase chain reaction. Chi-square tests were used for proportion differences and Student’s-t tests and variance analysis were used for testing differences between mean values. Odds ratio was used as a measure of association with a 95% confidence interval.

Results

The prevalence of PTB during the period of the study was 26% and the prevalence of CT among them was 13.9%. A total of 31.6% pregnant women were younger than 25 years old and women infected by CT were even younger than women not infected by CT (p = 0.022). Most of them (76.2%) were married or had a living partner, and CT infection was more frequent among the single ones (p = 0.018); 16.7% of women reported their first sexual intercourse under 14 years old. The causes of prematurity were maternal-fetal in 40.9%; rupture of the membranes in 29.7% and premature labor in 29.4%. In multivariate analysis, being married was a protective factor for infection [OR = 0.48 (95%CI:0.24–0.97)]. None of the other characteristics were associated with CT infection.

Conclusions

This study shows a high prevalence of CT infection among parturient who have preterm birth. This high prevalence highlight the need for defining screening strategies focused on young pregnant women in Brazil.     

The biomarkers of fetal growth in intrauterine growth retardation and large for gestational age cases: from adipocytokines to a metabolomic all-in-one tool

Adipose tissue is no longer considered as inert; the literature describes the role it plays in the production of many substances, such as adiponectin, visfatin, ghrelin, S100B, apelin, TNF, IL-6 and leptin. These molecules have specific roles in humans and their potential as biomarkers useful for identifying alterations related to intrauterine growth retardation and large for gestational age neonates is emerging. Infants born in such conditions have undergone metabolic changes, such as fetal hypo- or hyperinsulinemia, which may lead to development of dysmetabolic syndrome and other chronic diseases in adulthood. In this review, these biomarkers are analyzed specifically and it is discussed how metabolomics may be an advantageous tool for detection, discrimination and prediction of metabolic alterations and diseases. Thus, a holistic approach, such as metabolomics, could help the prevention and early diagnosis of metabolic syndrome.     

Isolation and characterization of a novel bladder cancer cell line: Inhibition by epidermal growth factor

Summary A novel continuous cell line, designated BC3c, was established from a surgical biopsy of an invasive solid transitional cell carcinoma of the bladder derived from an 82-yr-old Caucasian female. BC3c cells were near-triploid bearing multiple structural and numerical chromosome anomalies. The epithelial origin of the cancer cells was indicated by the expression of cytokeratins 8 and 19 as well as by the absence of mesenchymal markers. Polymerase chain reaction-restriction-fragment length polymorphisms and single-strand conformation polymorphism mutation detection assays did not reveal any mutations in H-ras codon 12 and K-ras codons 12 and 13. In addition, no mutation in specific hot-spot codons of the p53 gene and no accumulation of the p53 protein were observed. BC3c cells grew rapidly in vitro, even in the absence of exogenous growth factors, because they were found to stimulate their growth in an autocrine manner. BC3c cells were found to express the epidermal growth factor-receptor (EGF-r) abundantly, but in contrast to other established bladder cancer cell lines, human recombinant epidermal growth factor inhibited the cells’ proliferation in vitro. These features render the newly established bladder cancer cell line BC3c a useful tool for further experimentation.     

Infectious salmon anaemia virus (ISAV) isolated from the ISA disease outbreaks in Chile diverged from ISAV isolates from Norway around 1996 and was disseminated around 2005, based on surface glycoprotein gene sequences

Background

All viruses in the family Bunyaviridae possess a tripartite genome, consisting of a small, a medium, and a large RNA segment. Bunyaviruses therefore possess considerable evolutionary potential, attributable to both intramolecular changes and to genome segment reassortment. Hantaviruses (family Bunyaviridae, genus Hantavirus) are known to cause human hemorrhagic fever with renal syndrome or hantavirus pulmonary syndrome. The primary reservoir host of Sin Nombre virus is the deer mouse (Peromyscus maniculatus), which is widely distributed in North America. We investigated the prevalence of intramolecular changes and of genomic reassortment among Sin Nombre viruses detected in deer mice in three western states.

Methods

Portions of the Sin Nombre virus small (S) and medium (M) RNA segments were amplified by RT-PCR from kidney, lung, liver and spleen of seropositive peromyscine rodents, principally deer mice, collected in Colorado, New Mexico and Montana from 1995 to 2007. Both a 142 nucleotide (nt) amplicon of the M segment, encoding a portion of the G2 transmembrane glycoprotein, and a 751 nt amplicon of the S segment, encoding part of the nucleocapsid protein, were cloned and sequenced from 19 deer mice and from one brush mouse (P. boylii), S RNA but not M RNA from one deer mouse, and M RNA but not S RNA from another deer mouse.

Results

Two of 20 viruses were found to be reassortants. Within virus sequences from different rodents, the average rate of synonymous substitutions among all pair-wise comparisons (π_s) was 0.378 in the M segment and 0.312 in the S segment sequences. The replacement substitution rate (π_a) was 7.0 × 10^-4 in the M segment and 17.3 × 10^-4 in the S segment sequences. The low π_a relative to π_s suggests strong purifying selection and this was confirmed by a Fu and Li analysis. The absolute rate of molecular evolution of the M segment was 6.76 × 10^-3 substitutions/site/year. The absolute age of the M segment tree was estimated to be 37 years. In the S segment the rate of molecular evolution was 1.93 × 10^-3 substitutions/site/year and the absolute age of the tree was 106 years. Assuming that mice were infected with a single Sin Nombre virus genotype, phylogenetic analyses revealed that 10% (2/20) of viruses were reassortants, similar to the 14% (6/43) found in a previous report.

Conclusion

Age estimates from both segments suggest that Sin Nombre virus has evolved within the past 37–106 years. The rates of evolutionary changes reported here suggest that Sin Nombre virus M and S segment reassortment occurs frequently in nature.     

Lateglacial climate development in NW Romania — Comparative results from three quantitative pollen-based methods

This paper provides quantitative reconstructions of the Lateglacial changes in four climate parameters from two fine-resolution pollen profiles in the Gutaiului Mountains, NW Romania. Climate estimates are based on two modern analogue techniques (with and without considering vegetation types) and weighted averaging partial least squares regression (WA-PLS), giving evidence for several climatic fluctuations during the period from > 14,700 to 11,500 cal. yr BP. The comparative results of the two modern analogue techniques show consistent trends of climate changes that are also coherent at both sites, but these results appear to largely disagree compared with climate reconstruction provided by WA-PLS.The modern analogue techniques revealed four intervals with low temperatures: prior to 14,700 cal. yr BP, between 13,950 and 13,800; 13,400 and 13,200; and 12,700 and 11,700 cal. yr BP. The temperature declines were more pronounced for winter than for summer, suggesting an intensification of seasonality, which together with a drop in precipitation indicates an increase in continentality. The Younger Dryas is the most pronounced cooling phase with winter temperatures ~ 14–16 °C colder than modern conditions, annual and summer temperatures ~ 2–5 °C and ~ 2 °C, respectively below present ones. Precipitation was ~ 400–500 mm, half that of present. During the Bølling and Allerød, summer temperatures were close to modern values (13 to 17 °C), whereas winter (− 6 to − 12 °C) and annual temperatures (0.5 to 6 °C) as well as precipitation were (550 to 700 mm) lower, indicating more continental conditions compared to the present-day climate.     

Autoantigen discovery with a synthetic human peptidome

Immune responses targeting self-proteins (autoantigens) can lead to a variety of autoimmune diseases. Identification of these antigens is important for both diagnostic and therapeutic reasons. However, current approaches to characterize autoantigens have, in most cases, met only with limited success. Here we present a synthetic representation of the complete human proteome, the T7 peptidome phage display library (T7-Pep), and demonstrate its application to autoantigen discovery. T7-Pep is composed of >413,000 36-residue, overlapping peptides that cover all open reading frames in the human genome, and can be analyzed using high-throughput DNA sequencing. We developed a phage immunoprecipitation sequencing (PhIP-Seq) methodology to identify known and previously unreported autoantibodies contained in the spinal fluid of three individuals with paraneoplastic neurological syndromes. We also show how T7-Pep can be used more generally to identify peptide-protein interactions, suggesting the broader utility of our approach for proteomic research.