Biochemical functionality and recovery of hepatocytes after deep freezing storage

Summary The present study was undertaken to define the conditions for optimal cryopreservation of hepatocytes. Two different freezing procedures were analyzed: a slow freezing rate (SFR) (−2° C/min down to −30°C and then quick freezing to −196° C) and a fast freezing rate (FFR) (direct freezing of tubes to −196° C: −39° C/min). Cells were frozen in fetal bovine serum containing 10% Dimethyl sulfoxide (DMSO). After rapid thawing at 37° C, followed by dilution and removal of the cryoprotectant, cells were plated and several parameters were followed as criteria for optimal cryopreservation of cells. The FFR cells showed no apparent ultrastructural damage after 24 h of culture. Plating efficiency and spreading were similar as controls. Gluconeogenesis from pyruvate and fructose, tyrosine amino transferase induction by glucagon and dexamethasone, urea production, and plasma protein synthesis of FFR cells were similar to those found in control cultures. The FFR procedure, in comparison to the SFR method, seemed to render the best preserved hepatocytes. The financial support for this work was from Fondo de Investigaciones Sanitarias de la Seguridad Social, Grants 41/82 and 48/82.     

Evolutionary history of the European free-tailed bat,a tropical affinity species spanning across the Mediterranean Basin

The Mediterranean Basin is a global biodiversity hotspot, hosting a number of native species belonging to families that are found almost exclusively in tropical climates. Yet, whether or not these taxa were able to survive in the Mediterranean region during the Quaternary climatic oscillations remains unknown. Focusing on the European free-tailed bat (Tadarida teniotis), we aimed to (a) identify potential ancient populations and glacial refugia; (b) determine the post-glacial colonization routes across the Mediterranean; and (c) evaluate current population structure and demography. Mitochondrial and nuclear markers were used to understand T. teniotis evolutionary and demographic history. We show that T. teniotis is likely restricted to the Western Palearctic, with mitochondrial phylogeny suggesting a split between an Anatolian/Middle East clade and a European clade. Nuclear data pointed to three genetic populations, one of which is an isolated and highly differentiated group in the Canary Islands, another distributed across Iberia, Morocco, and France, and a third stretching from Italy to the east, with admixture following a pattern of isolation by distance. Evolutionary and demographic reconstruction supports a pre-Last Glacial Maximum (LGM) colonization of Italy and the Anatolian/Middle East, while the remaining populations were colonized from Italy after the Younger Dryas. We also found support for demographic expansion following the Iberian colonization. The results show that during the LGM T. teniotis persisted in Mediterranean refugia and has subsequently expanded to its current circum-Mediterranean range. Our findings raise questions regarding the physiological and ecological traits that enabled species with tropical affinities to survive in colder climates.     

966. CLAVIJA DOMINGENSIS

Clavija domingensis Urb. & Ekman was one of the many Haitian endemics that were described based on collections made by the great Swedish botanist Leonard Ekman between 1924 and 1928. The species is Critically Endangered sensu IUCN (criteria c2a(i); D) and it is currently the focus of conservation initiatives in Jardin Botanique des Cayes (Haiti), Jardín Botánico Nacional Dr. Rafael M. Moscoso (Dominican Republic), and Fairchild Tropical Botanic Garden (U.S.A.). Now known from only six localities from southern Haiti, each locality only represents a single individual. The species is illustrated based on plants grown in Fairchild Tropical Botanic Garden.     

Isolation and PCR amplification of genomic DNA from dried capsules of cardamon (Elettaria cardamomum M.)

Cardamom is an important spice, condiment and medicine, and international commodity. DNA-based molecular profiling will be aid in protecting the intellectual property rights of those who trade cardamom on the world market. Commercial cardamom has so far proven recalcitrant to traditional DNA extraction methods. In this paper we report a protocol for the isolation of amplifiable genomic DNA from traded cardamom. The method involves a modified CTAB (hexadecyltrimethylammonium bromide) extraction step, followed by a purification step to remove polysaccharides, proteins, and polyphenols, which are abundant in storage tissue such as cardamom capsules. The yield of DNA was 6–7 μg g⁻¹ tissue. Spectrophotometric and electrophoretic analysis indicated that the isolated DNA was highly pure and of high molecular weight. The isolated DNA could be amplified using different random decamer primers. The protocol has trade implications as it will help in the PCR-based characterisation of traded cardamom. This protocol can be further extended to develop Sequence Characterised Amplified Regions (SCAR) markers for profiling cardamoms.     

Genetics of schizophrenia and smoking: an approach to studying their comorbidity based on epidemiological findings

The association between schizophrenia and tobacco smoking has been described in more than 1,000 articles, many with inadequate methodology. The studies on this association can focus on: (1) current smoking, ever smoking or smoking cessation; (2) non-psychiatric controls or controls with severe mental illness (e.g., bipolar disorder); and (3) higher smoking frequency or greater usage in smokers. The association with the most potential for genetic studies is that between ever daily smoking and schizophrenia; it may reflect a shared genetic vulnerability. To reduce the number of false-positive genes, we propose a three-stage approach derived from epidemiological knowledge. In the first stage, only genetic variations associated with ever daily smoking that are simultaneously significant within the non-psychiatric controls, the bipolar disorder controls and the schizophrenia cases will be selected. Only those genetic variations that are simultaneously significant in the three hypothesis tests will be tested in the second stage, where the prevalence of the genes must be significantly higher in schizophrenia than in bipolar disorder, and significantly higher in bipolar disorder than in controls. The genes simultaneously significant in the second stage will be included in a third stage where the gene variations must be significantly more frequent in schizophrenia patients who did not start smoking daily until their 20s (late start) versus those who had an early start. Any genetic approach to psychiatric disorders may fail if attention is not given to comorbidity and epidemiological studies that suggest which comorbidities are likely to be explained by genetics and which are not. Our approach, which examines the results of epidemiological studies on comorbidities and then looks for genes that simultaneously satisfy epidemiologically suggested sets of hypotheses, may also apply to the study of other major illnesses.     

Adsorption of HSA, IgG and laminin-1 on model hydroxyapatite surfaces--effects of surface characteristics

Ellipsometry and mechanically assisted sodium dodecyl sulphate elution was utilized to study the adsorption of human serum albumin (HSA), human immunoglobulin G (IgG), and laminin-1, as well as competitive adsorption from a mixture of these proteins on spin-coated and sintered hydroxyapatite (HA) surfaces, respectively. The HA surfaces were characterized with respect to wettability and roughness by means of water contact angles and atomic force microscopy, respectively. Both surface types were hydrophilic, and the average roughness (Sa) and surface enlargement (Sdr) were lower for the sintered compared to the spin-coated HA surfaces. The adsorbed amounts on the sintered HA increased as follows: HSA < laminin-1 < IgG < the protein mixture. For the competitive adsorption experiments, the adsorbed fractions increased accordingly: HSA < laminin-1 < IgG on both types of HA substratum. However, a higher relative amount of HSA and laminin-1 and a lower relative amount of IgG was found on the spin-coated surfaces compared to the sintered surfaces. The effects observed could be ascribed to differences in surface roughness and chemical composition between the two types of HA substratum, and could have an influence on selection of future implant surface coatings.     

Exploration of the valproic acid binding site on histone deacetylase 8 using docking and molecular dynamic simulations

Epigenetic therapy is an important focus of research for drug development in the treatment of cancer. Valproic acid (VPA) is an HDAC inhibitor that has been evaluated in clinical studies. Despite its success in treating cancer, the mechanism of inhibition of VPA in HDAC is unknown. To this end, we have used docking and molecular dynamic simulations to investigate VPA binding to HDAC, employing both native and rebuilt 3-D structures. The results showed that VPA, via its carboxyl group, coordinates the Zn atom and other local residues (H141-142 and Y360) located at the catalytic site (CS) of HDAC. This causes electrostatic and hydrogen bonding interactions while having little interaction with the hydrophobic side chains, resulting in a low affinity. However, after several docking studies on different native HDAC 3-D structures and after using several snapshots from MD simulations, it became apparent that VPA bound with highest affinity at a site located at the acetyl-releasing channel, termed the hydrophobic active site channel (HASC). The affinity of VPA for HASC was due to its highly hydrophobic properties that allow VPA to take part in van der Waals interactions with Y18, I19, Y20, V25, R37, A38, V41, H42, I135 and W137, while VPA's carboxylate group has several hydrogen bonding interactions with the backbones of S138, I19, N136 and W137. MD simulations showed that the HASC door continuously opened and closed, which affected the affinity of VPA to the HASC, but the affinity toward the HASC was consistently higher than that obtained for the CS, suggesting that the HASC could be involved in the mechanism of inhibition.     

Genome-wide association study of multiple sclerosis confirms a novel locus at 5p13.1

Multiple Sclerosis (MS) is the most common progressive and disabling neurological condition affecting young adults in the world today. From a genetic point of view, MS is a complex disorder resulting from the combination of genetic and non-genetic factors. We aimed to identify previously unidentified loci conducting a new GWAS of Multiple Sclerosis (MS) in a sample of 296 MS cases and 801 controls from the Spanish population. Meta-analysis of our data in combination with previous GWAS was done. A total of 17 GWAS-significant SNPs, corresponding to three different loci were identified:HLA, IL2RA, and 5p13.1. All three have been previously reported as GWAS-significant. We confirmed our observation in 5p13.1 for rs9292777 using two additional independent Spanish samples to make a total of 4912 MS cases and 7498 controls (ORpooled = 0.84; 95%CI: 0.80-0.89; p = 1.36 × 10-9). This SNP differs from the one reported within this locus in a recent GWAS. Although it is unclear whether both signals are tapping the same genetic association, it seems clear that this locus plays an important role in the pathogenesis of MS.     

A novel human polycomb binding site acts as a functional polycomb response element in Drosophila

Polycomb group (PcG) proteins are key chromatin regulators implicated in multiple processes including embryonic development, tissue homeostasis, genomic imprinting, X-chromosome inactivation, and germ cell differentiation. The PcG proteins recognize target genomic loci through cis DNA sequences known as Polycomb Response Elements (PREs), which are well characterized in Drosophila. However, mammalian PREs have been elusive until two groups reported putative mammalian PREs recently. Consistent with the existence of mammalian PREs, here we report the identification and characterization of a potential PRE from human T cells. The putative human PRE has enriched binding of PcG proteins, and such binding is dependent on a key PcG component SUZ12. We demonstrate that the putative human PRE carries both genetic and molecular features of Drosophila PRE in transgenic flies, implying that not only the trans PcG proteins but also certain features of the cis PREs are conserved between mammals and Drosophila.     

Direct measurements of the kinematics and dynamics of bat flight

Experimental measurements and analysis of the flight of bats are presented, including kinematic analysis of high-speed stereo videography of straight and turning flight, and measurements of the wake velocity field behind the bat. The kinematic data reveal that, at relatively slow flight speeds, wing motion is quite complex, including a sharp retraction of the wing during the upstroke and a broad sweep of the partially extended wing during the downstroke. The data also indicate that the flight speed and elevation are not constant, but oscillate in synchrony with both the horizontal and vertical movements of the wing. PIV measurements in the transverse (Trefftz) plane of the wake indicate a complex 'wake vortex' structure dominated by a strong wing tip vortex shed from the wing tip during the downstroke and either the wing tip or a more proximal joint during the upstroke. Data synthesis of several discrete realizations suggests a 'cartoon' of the wake structure during the entire wing beat cycle. Considerable work remains to be done to confirm and amplify these results.