bluuebunny/crossref_metadata_2025 huggingface dataset:
FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment
Authors: Kunath Tilo , Saba-El-Leil Marc K. , Almousailleakh Marwa , Wray Jason , Meloche Sylvain , Smith Austin
Published: July 2007

Pluripotent embryonic stem (ES) cells must select between alternative fates of self-replication and lineage commitment during continuous proliferation. Here, we delineate the role of autocrine production of fibroblast growth factor 4 (Fgf4) and associated activation of the Erk1/2 (Mapk3/1) signalling cascade. Fgf4 is the major stimulus activating Erk in mouse ES cells. Interference with FGF or Erk activity using chemical inhibitors or genetic ablations does not impede propagation of undifferentiated ES cells. Instead,such manipulations restrict the ability of ES cells to commit to differentiation. ES cells lacking Fgf4 or treated with FGF receptor inhibitors resist neural and mesodermal induction, and are refractory to BMP-induced non-neural differentiation. Lineage commitment potential of Fgf4-null cells is restored by provision of FGF protein. Thus, FGF enables rather than antagonises the differentiation activity of BMP. The key downstream role of Erk signalling is revealed by examination of Erk2-null ES cells,which fail to undergo either neural or mesodermal differentiation in adherent culture, and retain expression of pluripotency markers Oct4, Nanog and Rex1. These findings establish that Fgf4 stimulation of Erk1/2 is an autoinductive stimulus for naïve ES cells to exit the self-renewal programme. We propose that the Erk cascade directs transition to a state that is responsive to inductive cues for germ layer segregation. Consideration of Erk signalling as a primary trigger that potentiates lineage commitment provides a context for reconciling disparate views on the contribution of FGF and BMP pathways during germ layer specification in vertebrate embryos.
Computational quantum mechanics: An underutilized tool in thermodynamics
Authors: Sandler Stanley I. , Castier Marcelo
Published: July 2007

In this paper, we highlight the various ways computational quantum mechanics (QM) can be used in applied thermodynamics. We start with the most rigorous procedures of calculating the interactions between molecules that can then be used in simulation and progress, in steps, to less rigorous but easily used methods, including the very successful continuum solvation models.
Bond-dissociation enthalpies in the gas phase and in organic solvents: Making ends meet
Authors: Borges dos Santos Rui M. , Costa Cabral Benedito J. , Martinho Simões José A.
Published: July 2007

Solvent effects are responsible for the difference between gas- and solution-phase bond-dissociation enthalpies (BDEs), and are thus crucial for understanding reactivity in solution. While solvation effects can be negligible (e.g., in reactions involving carbon-centered radicals), they may be rather significant (e.g., when oxygen-centered radicals are formed). This paper reviews a number of models which have been proposed to deal with the difference between the solvation energetics of a radical and its parent molecule. It is concluded that the radical-solvent interaction may be larger than previously anticipated.
Specific Magnetic Bead–Based Capture of Genomic DNA from Clinical Samples: Application to the Detection of Group B Streptococci in Vaginal/Anal Swabs
Authors: Parham Nicholas J , Picard François J , Peytavi Régis , Gagnon Martin , Seyrig Grégoire , Gagné Pier-Ann , Boissinot Maurice , Bergeron Michel G
Published: July 2007

Background: Group B streptococci (GBS) are a leading cause of sepsis and meningitis in newborns. We previously developed a rapid diagnostic system for GBS detection from vaginal/anal samples obtained from pregnant women during delivery. To facilitate the adaptation of this method for point-of-care testing, we have developed a specific and efficient GBS DNA capture method that is compatible with both PCR and nonamplification detection technologies.

Methods: Superparamagnetic beads were functionalized with oligonucleotide capture probes of different lengths and used to capture GBS genomic DNA (gDNA). A rapid extraction procedure was used to provide DNA from GBS cultures or vaginal/anal samples with added GBS. Hybridization reactions consisting of functionalized beads and target DNA in 30 μL of hybridization buffer were performed for 1 h at room temperature, followed by washing and resuspension in water. Captured DNA was then detected using quantitative PCR.

Results: A 25-mer capture probe allowed detection of 1000 genome copies of purified GBS DNA. The ability to detect GBS was improved by use of a 50-mer (100 copies) and a 70-mer capture probe (10 copies). Detection of approximately 1250 CFU/mL was achieved for diluted GBS broth culture and for vaginal/anal swab samples with added GBS.

Conclusion: Oligonucleotide-functionalized superparamagnetic microbeads efficiently capture GBS gDNA from both bacterial cultures and vaginal/anal samples with added GBS. Efficiency of gDNA capture increases with oligonucleotide length. This technology could be combined with sample preparation and detection technologies in a microfluidic system to allow point-of-care testing for GBS.
War, wounds and women: The Spanish Civil War in Victor Erice's El espritu de la colmena and David Trueba's Soldados de Salamina
Authors: Camino Mercedes M
Published: July 2007

The dreams and losses entailed by the Spanish Civil War have been captured in many films. In Spain, however, the war is often looked at from the vantage point of its aftermath, such as in Victor Erice's classic,El espritu de la colmena (The Spirit of the Beehive, 1973), and, more recently, in David Trueba's adaptation of Javier Cercas' novel,Soldados de Salamina (Soldiers of Salamis, 2003). This article scrutinises the intimate relationship between the haunting presence of the war in these films and their use of a female protagonist to signify that past. The myth of the Spanish Civil War, I argue, is reinforced by the way in which audiences see it through the innocent eyes of Erice's Ana or those of Trueba's Lola. These females become, for us, true speakers of the war and its effects, thus contributing to the reification of a historical past that has been made attractive precisely by the sadness that it conveys.
Effect of bleomycin and cisplatin on the expression profile ofSRA1, a novel member of pre-mRNA splicing factors, in HL-60 human promyelocytic leukemia cells
Authors: Katsarou Maria E. , Thomadaki Hellinida , Katsaros Nikos , Scorilas Andreas
Published: July 2007

Recently, a new member of the human SR (Ser/Arg-rich) superfamily of pre-mRNA splicing factors, SRA1 (SR-A1), has been discovered and cloned by members of our group, the gene for which was found to be overexpessed in a series of human tumors. In the present study, we investigated the significance of alterations at the mRNA expression levels of theSRA1gene after treatment of HL-60 human promyelocytic leukemia cells with the anticancer drugs cisplatin and bleomycin. The kinetics of apoptosis and cell toxicity were investigated by DNA laddering and the MTT and trypan blue assays, respectively. Total RNA was extracted and cDNA was prepared by reverse transcription. The splicing-related genesSRA1andSC35, as well as the apoptosis-related geneBCL2(Bcl-2), were amplified by PCR using gene-specific primers. The results showed that mRNA levels ofSRA1were up-regulated upon treatment with the antibiotic bleomycin, whereas they were down-regulated by treatment of HL-60 human promyelocytic leukemia cells with cisplatin. Our results support the hypothesis that mRNA expression analysis ofSRA1may serve as a new prospective molecular marker, playing an important role in chemotherapy outcome in human leukemia.
In vivo labeling with stable isotopes as a tool for the identification of unidentified peaks in the metabolome analysis of Corynebacterium glutamicum by GC/MS
Authors: Herebian Diran , Küpper Uta , Schomburg Dietmar , Marner Franz-Josef
Published: July 2007

Of the hyphenated techniques used for metabolic profiling of cell and tissue extracts, GC/MS is in some ways advantageous as it allows the simultaneous fingerprinting of chemically very different metabolites, and the electron impact mass spectra recorded in many cases lead to unambiguous identification of the compounds. However, prior to chromatography, the hydrophilic substances of the cell extracts have to be converted to vaporizable derivatives, the mass spectra of which often are not known or not listed in the available spectral libraries, even if they are derived from simple biochemicals. Thus, numerous chromatographic peaks remain as yet unidentified. Attempts to identify these peaks afford the acquisition of more data on these compounds. The value of in vivo labeling of metabolites with 13C and 15N for this purpose is described.
Cleavage of the myristoylated alanine-rich C kinase substrate (MARCKS) by cysteine cathepsins in cells and tissues of stefin B-deficient mice
Authors: Kopitar-Jerala Nataša , Turk Boris
Published: July 2007

The myristoylated alanine-rich C kinase substrate (MARCKS) is a substrate of protein kinase C (PKC). Besides regulation at the level of gene transcription, MARCKS concentrations within the cell are also regulated by proteolytic cleavage by cathepsins and calpains, which are cysteine proteinases. Stefin B (cystatin B) is an endogenous inhibitor of lysosomal cysteine cathepsins, but not calpains. We have observed increased cleavage of MARCKS in brain and macrophages, but not in liver and kidney extracts of stefin B-deficient mice compared to wild-type mice. Processing of cathepsin B was unaltered in the brain of stefin B-deficient mice and we conclude that increased cleavage of MARCKS could be attributed to the lack of inhibitor.
Involvement of Kupffer cell-dependent signaling in T3-induced hepatocyte proliferationin vivo
Authors: Fernández Virginia , Reyes Solange , Bravo Sergio , Sepúlveda Rodrigo , Romanque Pamela , Santander Gonzalo , Castillo Iván , Varela Patricia , Tapia Gladys , Videla Luis A.
Published: July 2007

Thyroid hormone-induced calorigenesis triggers liver oxidative stress with concomitant TNF-α production by Kupffer cells and up-regulation of gene expression. Considering that cyclin-dependent kinase-2 (CDK-2) performs essential functions for cellular proliferation, our aim was to test the hypothesis thatl-3,3′,5-triiodothyronine (T3) stimulates liver cell proliferation by upstream mechanisms involving CDK-2 expression dependent on Kupffer cell signaling. T3administration induced a calorigenic response at 60–70 h after treatment, with increased TNF-α generation and hepatic oxidative stress status, as shown by enhanced protein carbonyls and decreased glutathione content compared to controls. In this time interval, liver c-jun N-terminal kinase (JNK) phosphorylation, activator protein-1 (AP-1) DNA binding, and CDK-2 expression were enhanced, with concomitantly higher levels of the proliferation markers Ki-67 and proliferating cell nuclear antigen. These changes are abolished by administration of the Kupffer cell inactivator gadolinium chloride prior to T3treatment. We conclude that T3administration triggers liver CDK-2 expression and cellular proliferation through a cascade associated with Kupffer cell-dependent TNF-α generation, JNK phosphorylation, and AP-1 activation. Since CDK-2 promotes phase S progression within the cell cycle, this response may constitute a major mechanism involved in T3-induced liver preconditioning to ischemia/reperfusion injury.
Engineering high-speed allosteric hammerhead ribozymes
Authors: Link Kristian H. , Guo Lixia , Ames Tyler D. , Yen Laising , Mulligan Richard C. , Breaker Ronald R.
Published: July 2007

Full-length hammerhead ribozymes were subjected to in vitro selection to identify variants that are allosterically regulated by theophylline in the presence of a physiologically relevant concentration of Mg2+. The population of allosteric ribozymes resulting from 15 rounds of in vitro selection yielded variants with observed rate constants (k
obs) as high as 8 min-1 in the presence of theophylline and maximal k
obs increases of up to 285-fold compared to rate constants measured in the absence of effector. The selected ribozymes have kinetic characteristics that are predicted to be sufficient for cellular gene control applications, but do not exhibit any activity in reporter gene assays. The inability of the engineered RNAs to control gene expression suggests that the in vitro and in vivo folding pathways of the RNAs are different. These results provide several key pieces of information that will aid in future efforts to engineer allosteric ribozymes for gene control applications.
Oestrogen and progesterone receptors in the marmoset endometrium: changes during the ovulatory cycle, early pregnancy and after inhibition of vascular endothelial growth factor, GnRH or ovariectomy
Authors: Silvestri Audrey , Fraser Hamish M
Published: July 2007

Marmosets are widely used, but detailed studies on localisation of endometrial oestrogen receptors α and β (ERα and ERβ ), and the progesterone receptor (PR) are lacking. These receptors were localised and semi-quantitatively analysed throughout the ovulatory cycle, weeks 2, 3 and 4 of pregnancy and after treatment with GnRH antagonist, vascular endothelial growth factor (VEGF) Trap or ovariectomy. The PR in epithelial cells increased markedly between the mid- and late proliferative phases before declining in the mid-secretory phase and pregnancy. PR in stromal cells was present throughout the cycle and levels were maintained in pregnancy. ERα was present at the mid-proliferative phase and increased in glands at the late proliferative and early secretory phases, before declining at the late secretory phase and week 4 of pregnancy. Stromal ERα showed a similar trend, but decreased earlier, by the mid-secretory phase. ERβ was highly expressed in epithelial cells throughout the cycle and in pregnancy. In stroma, increases in ERβ expression were observed at the late proliferative phase with the staining index decreasing by half as the secretory phase progressed and in pregnancy. GnRH antagonist, VEGF Trap or ovariectomy caused significant reductions in PR and ERβ expression, but not in ERα when compared with the late proliferative phase of the normal cycle. Endothelial cells expressed ERβ , but not ERα or PR. It is concluded that the steroid receptor profile in the marmoset endometrium is generally similar to the human and should provide a useful model for studies on hormonal manipulation of the endometrium.
The effects of oestrogen receptors α and β on testicular cell number and steroidogenesis in mice
Authors: Gould M L , Hurst P R , Nicholson H D
Published: July 2007

Oestrogen plays an important role in testicular function. This study used mice null for oestrogen receptor α (ERα) or β (ERβ) to investigate which receptor mediates the effects of oestrogen within the testis. Groups of ERα knockout mice (αERKO) and ERβ knockout mice (βERKO) and wild-type littermates (n=5–8) were killed at 11 weeks post partum. One testis was fixed in Bouin’s fluid for stereology and the other frozen for testosterone measurement. Trunk blood was collected for testosterone RIA. The optical disector combined with the fractionator methodology was used to estimate Leydig, Sertoli and germ cell numbers. At all times, the knockout animals were compared with their wild-type littermates. The physical disector quantified cells stained immunohistochemically for the apoptotic marker active caspase-3 and Hoechst staining was used to identify nuclear fragmentation. The mean Leydig cell volume was measured using the point sampled intercept method. The Leydig cell number per testis was significantly increased in βERKO mice but not in αERKO mice. Plasma and testicular testosterone concentrations were increased in αERKO mice but no changes were observed in βERKO mice. Hypertrophic Leydig cell changes were observed in αERKO mice, and a decreased mean cell volume was seen in βERKO mice. No difference in Sertoli cell number per testis was observed in any of the groups. The spermatogonial cell number per testis was increased in βERKO mice. Immunohistochemistry identified increased numbers of active caspase-3-labelled germ cells per testis in αERKO mice but not βERKO mice. Hoechst staining supported these findings. There was significant germ cell loss in αERKO mice. This study suggests that ERβ may be involved in regulation of Leydig cell proliferation and testosterone production in the adult mouse testis.
Production of cloned horse foals using roscovitine-treated donor cells and activation with sperm extract and/or ionomycin
Authors: Hinrichs K , Choi Y H , Varner D D , Hartman D L
Published: July 2007

We evaluated the effect of different activation treatments on the production of blastocysts and foals by nuclear transfer. Donor cells were prepared using roscovitine treatment, which has previously been associated with increased production of viable offspring. All activation treatments were followed by culture in 6-dimethylaminopurine (6-DMAP) for 4 h. In experiment 1, blastocyst production after activation by injection of sperm extract followed by treatment with ionomycin was significantly higher than that for activation with a serial treatment of ionomycin, 6-DMAP, and ionomycin (12.5 vs 2.8%; P < 0.05) and tended to be higher than that for injection of sperm extract alone (3.4%; P = 0.07). In experiment 2, there were no significant differences in blastocyst development among treatments with ionomycin once or twice, sperm extract then ionomycin, or ionomycin then sperm extract (range 4.6–7.3%). Overall, transfer of 26 blastocysts resulted in 16 pregnancies (62%) and 9 live foals (35% of transferred embryos). Treatment with sperm extract followed by ionomycin produced a live foal rate per embryo transferred of 5/10 (50%). One foal died of pneumonia 48 h post partum and one foal died at 1 week of age after complications during induction of anesthesia; the remaining seven foals are currently 10–14 months of age.
Association of three single nucleotide polymorphisms of the E-cadherin gene with endometriosis in a Chinese population
Authors: Shan Kang , Xiao-Wei Ma , Na Wang , Xiu-Feng Zhang , Deng-Gui Wen , Wei Guo , Zheng-Mao Zhang , Yan Li
Published: July 2007

Endometriosis, one of the most frequent diseases in gynecology, is a benign but invasive and metastatic disease. The altered expression of E-cadherin may play an important role in developing endometriosis. In this paper, we discuss the association of three single nucleotide polymorphisms (SNPs) on the E-cadherin gene and risk of endometriosis. We examined the genotype frequency of three polymorphisms in 152 endometriosis patients and 189 control women. There was a significant difference in the frequency of the E-cadherin 3′-UTR C → T genotypes between endometriosis and controls (P = 0.01). The frequency of the C allele in patients (71.1%) was significantly higher than in the controls (63.8%; P = 0.04). When compared with the T/T + T/C genotypes, the C/C genotype had a significantly increased susceptibility to endometriosis, with an adjusted odds ratio of 1.79 (95% confidence interval = 1.17–2.76). No significant difference was found between endometriosis and control women on two polymorphisms (−160 C → A, −347 G → GA) at the gene promoter region of E-cadherin. The −160 C → A and −347 G → GA polymorphisms displayed linkage disequilibrium (D′ = 0.999). The −160 A/−347 GA haplotype was only detected in endometriosis patients (2%). These data show a relation between the E-cadherin 3′-UTR C → T polymorphism, the −160 A/−347 GA haplotype of two promoter polymorphisms and risk of endometriosis, suggesting a potential role in endometriosis development, at least in North Chinese women.
Analysis of programmed cell death in mouse fetal oocytes
Authors: Lobascio A M , Klinger F G , Scaldaferri M L , Farini D , De Felici M
Published: July 2007

We report a short-term culture system that allowsto define novel characteristic of programmed cell death (PCD) in fetal oocytes and to underscore newaspects of this process. Mouse fetal oocytes culturedin conditions allowingmeiotic prophase I progression underwent apoptotic degeneration waves as revealed by TUNEL staining. TEM observations revealed recurrent atypical apoptotic morphologies characterized by the absence of chromatin margination and nuclear fragmentation; oocytes with autophagic and necrotic features were also observed. Further characterization of oocyte death evidenced DNA ladder, Annexin V binding, PARP cleavage, and usually caspase activation (namely caspase-2). In the aim to modulate the oocyte death process, we found that the addition to the culture medium of the pancaspase inhibitors Z-VAD orcaspase-2-specific inhibitor Z-VDVAD resulted in a partial and transient prevention of this process. Oocyte death was significantly reduced by the antioxidant agent NAC and partly prevented by KL and IGF-I growth factors. Finally, oocyte apoptosis was reduced by calpain inhibitor I and increased by rapamycin after prolonged culture.

These results support the notion that fetal oocytes undergo degeneration mostly by apoptosis. This process is, however, often morphologically atypical and encompasses other forms of cell death including caspase-independent apoptosis and autophagia. The observation that oocyte death occurs mainly at certain stages of meiosis and can only be attenuated by typical anti-apoptotic treatments favors the notion that it is controlled at least in part by stage-specific oocyte-autonomous meiotic checkpoints and when activated is little amenable to inhibition being the oocyte able to switch back and forth among different death pathways.
Economic Analysis of Triptan Therapy for Acute Migraine: A Medicaid Perspective
Authors: Mullins C. Daniel , Subedi Prasun R. , Healey Paul J. , Sanchez Robert J.
Published: July 2007

Study Objective. To compare triptan therapies for migraine in terms of the cost to treat 100 migraine attacks and the cost per successfully treated patient (cost/success), by analyzing utilization and reimbursement data from state Medicaid programs.

Design. Pharmacoeconomic analysis.

Data Source. Clinical efficacy data were obtained from a previously published meta‐analysis for 11 triptan‐dose combinations: almotriptan 12.5 mg; eletriptan 20 and 40 mg; naratriptan 2.5 mg; rizatriptan 5 and 10 mg; sumatriptan 25, 50, and 100 mg; and zolmitriptan 2.5 and 5 mg. Triptan reimbursement data were obtained from the Medicaid Drug Rebate Program of seven geographically dispersed states (Florida, Georgia, Illinois, North Carolina, Ohio, Wisconsin, and West Virginia).

Measurements and Main Results. Efficacy measures were derived based on data from the published meta‐analysis that evaluated headache pain status at 2 and 24 hours after triptan dosing. Reimbursement data for the triptans were applied to a previously developed model of migraine treatment outcomes to calculate the cost to treat 100 migraine attacks and the cost/success. Sensitivity analysis around dosing assumptions was conducted to assess robustness of estimates. Across the seven states, the two treatments associated with the lowest cost to treat 100 migraine attacks were eletriptan 20 mg (range $1549‐$1658) and eletriptan 40 mg ($1578‐$1661). Naratriptan 2.5 mg (range $1734‐$2018), sumatriptan 25 mg ($1853–1954), and zolmitriptan 5 mg ($1854‐$1960) were associated with the highest cost to treat 100 migraine attacks. Eletriptan 40 mg was associated with the lowest cost/success (range $57.03‐$60.05); naratriptan 2.5 mg ($99.39‐$115.65), sumatriptan 25 mg ($107.11‐$112.93), and rizatriptan 5 mg ($99.41‐$111.25) were associated with the highest cost/success values. Changes in dosing assumptions did not significantly change the rank ordering of triptans across either economic end point.

Conclusions. Eletriptan 20‐ and 40‐mg doses were shown to be associated with the lowest cost to treat 100 migraine attacks and the lowest cost/success in both the baseline and sensitivity analyses. These findings are consistent with results of similar economic analyses that compared multiple triptan therapies.
Benzocaine‐Induced Methemoglobinemia in Two Patients: Interdisciplinary Collaboration, Management, and Near Misses
Authors: Throm Melinda J. , Stevens Margie Dale , Hansen Carol
Published: July 2007

Methemoglobin, a form of hemoglobin that does not bind oxygen, is produced when iron in red blood cells is oxidized from the ferrous state to the ferric state. Methemoglobinemia develops in the presence of oxidizing agents, such as benzocaine‐containing topical anesthetic sprays, and it is characterized by cyanosis. If untreated, methemoglobinemia may prove lethal. We describe two patients who developed methemoglobinemia after they were administered benzocaine‐containing topical anesthetic sprays. Using the Naranjo adverse drug reaction probability scale, the relationship between the administration of the benzocaine‐containing spray and the development of methemoglobinemia was probable (score of 7) in both patients. Collaboration among health care providers is necessary to efficiently recognize, treat, and manage this condition.
Injectable Mesenchymal Stem Cell Therapy for Large Cartilage Defects—A Porcine Model
Authors: Lee Kevin B.L. , Hui James H.P. , Song Im Chim , Ardany Lenny , Lee Eng Hin
Published: July 2007

Current techniques in biological resurfacing of cartilage defects require an open arthrotomy or arthroscopy and involve the direct transplantation of isolated cells and/or scaffolds or whole tissue grafts with chondrogenic potential onto the cartilage defect. Our study investigates the possibility of direct intra-articular injection of mesenchymal stem cells suspended in hyaluronic acid (HA) as an alternative to the much more invasive methods currently available. A partial-thickness (without penetration of the subchondral bone) cartilage defect was created in the medial femoral condyle of an adult minipig. Mesenchymal stem cells from the iliac crest marrow of the same pig harvested in a separate procedure and suspended in 2 milliliters of hylan G-F 20 (Synvisc) were injected intra-articularly after the creation of the defect. This was followed by two more injections of hylan G-F 20 (HA) at weekly intervals. Either saline or HA was injected into the knees of the controls. The pigs were sacrificed at 6 and 12 weeks for morphological and histological analysis. The cell-treated groups showed improved cartilage healing both histologically and morphologically at 6 and 12 weeks compared with both controls. The use of intra-articular injections of mesenchymal stem cells suspended in HA is a viable option for treating large cartilage defects. This would be further explored in clinical trials.

Disclosure of potential conflicts of interest is found at the end of this article.
Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels and T-Type Calcium Channels Confer Automaticity of Embryonic Stem Cell-Derived Cardiomyocytes
Authors: Yanagi Kentoku , Takano Makoto , Narazaki Genta , Uosaki Hideki , Hoshino Takuhiro , Ishii Takahiro , Misaki Takurou , Yamashita Jun K.
Published: July 2007

Regeneration of cardiac pacemakers is an important target of cardiac regeneration. Previously, we developed a novel embryonic stem (ES) cell differentiation system that could trace cardiovascular differentiation processes at the cellular level. In the present study, we examine expressions and functions of ion channels in ES cell-derived cardiomyocytes during their differentiation and identify ion channels that confer their automaticity. ES cell-derived Flk1+ mesoderm cells give rise to spontaneously beating cardiomyocytes on OP9 stroma cells. Spontaneously beating colonies observed at day 9.5 of Flk1+ cell culture (Flk-d9.5) were significantly decreased at Flk-d23.5. Expressions of ion channels in pacemaker cells hyperpolarization-activated cyclic nucleotide-gated (HCN)1 and -4 and voltage-gated calcium channel (Cav)3.1 and -3.2 were significantly decreased in purified cardiomyocytes at Flk-d23.5 compared with at Flk-d9.5, whereas expression of an atrial and ventricular ion channel, inward rectifier potassium channel (Kir)2.1, did not change. Blockade of HCNs and Cav ion channels significantly inhibited beating rates of cardiomyocyte colonies. Electrophysiological studies demonstrated that spontaneously beating cardiomyocytes at Flk-d9.5 showed almost similar features to those of the native mouse sinoatrial node except for relatively deep maximal diastolic potential and faster maximal upstroke velocity. Although ∼60% of myocytes at Flk-d23.5 revealed almost the same properties as those at Flk-d9.5, ∼40% of myocytes showed loss of HCN and decreased Cav3 currents and ceased spontaneous beating, with no remarkable increase of Kir2.1. Thus, HCN and Cav3 ion channels should be responsible for the maintenance of automaticity in ES cell-derived cardiomyocytes. Controlled regulation of these ion channels should be required to generate complete biological pacemakers.

Disclosure of potential conflicts of interest is found at the end of this article.
Concise Review: Mesenchymal Stem Cells: Their Phenotype, Differentiation Capacity, Immunological Features, and Potential for Homing
Authors: Chamberlain Giselle , Fox James , Ashton Brian , Middleton Jim
Published: July 2007

MSCs are nonhematopoietic stromal cells that are capable of differentiating into, and contribute to the regeneration of, mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, and adipose. MSCs are rare in bone marrow, representing ∼1 in 10,000 nucleated cells. Although not immortal, they have the ability to expand manyfold in culture while retaining their growth and multilineage potential. MSCs are identified by the expression of many molecules including CD105 (SH2) and CD73 (SH3/4) and are negative for the hematopoietic markers CD34, CD45, and CD14. The properties of MSCs make these cells potentially ideal candidates for tissue engineering. It has been shown that MSCs, when transplanted systemically, are able to migrate to sites of injury in animals, suggesting that MSCs possess migratory capacity. However, the mechanisms underlying the migration of these cells remain unclear. Chemokine receptors and their ligands and adhesion molecules play an important role in tissue-specific homing of leukocytes and have also been implicated in trafficking of hematopoietic precursors into and through tissue. Several studies have reported the functional expression of various chemokine receptors and adhesion molecules on human MSCs. Harnessing the migratory potential of MSCs by modulating their chemokine-chemokine receptor interactions may be a powerful way to increase their ability to correct inherited disorders of mesenchymal tissues or facilitate tissue repair in vivo. The current review describes what is known about MSCs and their capacity to home to tissues together with the associated molecular mechanisms involving chemokine receptors and adhesion molecules.

Disclosure of potential conflicts of interest is found at the end of this article.
HIERARCHICAL SPATIAL MODELS OF ABUNDANCE AND OCCURRENCE FROM IMPERFECT SURVEY DATA
Authors: Royle J. Andrew , Kéry Marc , Gautier Roland , Schmid Hans
Published: July 2007

Many estimation and inference problems arising from large‐scale animal surveys are focused on developing an understanding of patterns in abundance or occurrence of a species based on spatially referenced count data. One fundamental challenge, then, is that it is generally not feasible to completely enumerate (“census”) all individuals present in each sample unit. This observation bias may consist of several components, including spatial coverage bias (not all individuals in the population are exposed to sampling) and detection bias (exposed individuals may go undetected). Thus, observations are biased for the state variable (abundance, occupancy) that is the object of inference. Moreover, data are often sparse for most observation locations, requiring consideration of methods for spatially aggregating or otherwise combining sparse data among sample units. The development of methods that unify spatial statistical models with models accommodating non‐detection is necessary to resolve important spatial inference problems based on animal survey data.

In this paper, we develop a novel hierarchical spatial model for estimation of abundance and occurrence from survey data wherein detection is imperfect. Our application is focused on spatial inference problems in the Swiss Survey of Common Breeding Birds. The observation model for the survey data is specified conditional on the unknown quadrat population size,N(s). We augment the observation model with a spatial process model forN(s), describing the spatial variation in abundance of the species. The model includes explicit sources of variation in habitat structure (forest, elevation) and latent variation in the form of a correlated spatial process. This provides a model‐based framework for combining the spatially referenced samples while at the same time yielding a unified treatment of estimation problems involving both abundance and occurrence.

We provide a Bayesian framework for analysis and prediction based on the integrated likelihood, and we use the model to obtain estimates of abundance and occurrence maps for the European Jay (Garrulus glandarius), a widespread, elusive, forest bird. The naive national abundance estimate ignoring imperfect detection and incomplete quadrat coverage was 77 766 territories. Accounting for imperfect detection added approximately 18 000 territories, and adjusting for coverage bias added another 131 000 territories to yield a fully corrected estimate of the national total of about 227 000 territories. This is approximately three times as high as previous estimates that assume every territory is detected in each quadrat.
CLIMATE AND PICEA MARIANA SEED MATURATION RELATIONSHIPS: A MULTI‐SCALE PERSPECTIVE
Authors: Meunier C. , Sirois L. , Bégin Y.
Published: July 2007

One of the most important ways by which northern forests will respond to anticipated climate change is through variations in seed maturation. In this study, the relationship between growing degree‐days (DD) >5°C and seed maturity was evaluated at three spatial scales. At the continental scale, the development of female gametophytes and embryos was evaluated as a function of the heat sums obtained from 11 sites distributed across the Canadian range of black spruce. At the regional scale, cone size and the percentage of germinated seeds formed in 1998, 1999, and 2000 were analyzed from seven sites situated along a latitudinal gradient in northern Quebec. At the local scale, cones were collected along perilacustral and insular transects according to their exposure to large water bodies, and from 10 islands located within a 2835‐km2 hydroelectric reservoir. Our results confirm the 800–940 DD thermal sum threshold necessary for the complete maturation of black spruce embryos at several populations distributed across the total range of the species. Along the regional south‐to‐north climatic gradient, the percentage of germination can be predicted by a sigmoid function of a thermal sum (y = 2.8 + 25.1/[1 + e(x−896.6)/84.6]; r2 = 0.85, P < 0.0001) that attains a plateau at around 800–940 DD. Once the 800–940 DD threshold is attained, variations in the percentage of seed germination are mainly associated with inter‐tree differences and local site factors (thickness of organic matter, tree density, tree height, tree age, and fetch). In the springtime, cold enclaves are created by the presence of the hydroelectric reservoir. The seed germination percentages in these enclaves varied from 0.6% ± 0.7% to 14.9% ± 19.1% (mean ± SD) according to the site (compared to 22.7% ± 15.1% for a site not exposed to the reservoir), which was equivalent to the germination percentages for sites at latitudes 1–3° farther north. These data suggest that the potential for black spruce regeneration increases strongly beyond the 800 DD isotherm, which evokes the possibility that subarctic open forests may become more dense under the current anticipated climate changes.
T-Cell Responses to Islet Antigens Improves Detection of Autoimmune Diabetes and Identifies Patients With More Severe β-Cell Lesions in Phenotypic Type 2 Diabetes
Authors: Goel Amit , Chiu Harvey , Felton Jamie , Palmer Jerry P. , Brooks-Worrell Barbara
Published: July 2007

Latent autoimmune diabetes in adults or type 1.5 diabetes is considered to be a T-cell–mediated autoimmune disease. However, identification of patients is based commonly on autoantibody (Ab) detection. To determine whether measuring T-cell reactivity to islet proteins compared with measuring Abs improves detection of autoimmune diabetes and how β-cell function correlates with T-cell reactivity compared with Ab positivity, we assessed the T-cell proliferative responses and Ab responses (islet cell autoantibodies, insulin autoantibodies, insulinoma-associated protein-2 autoantibodies, and GAD Abs) to islet proteins of 36 phenotypic type 2 diabetic patients. To be considered Ab+ or T-cell+, patients were required to be positive for a minimum of two consecutive time points. β-Cell function was measured with fasting and glucagon-stimulated C-peptide. Independent of T-cell reactivity, Ab+ and Ab− patients had comparable fasting and glucagon-stimulated C-peptide. Independent of Ab status, T-cell+ patients demonstrated significantly lower glucagon-stimulated (P < 0.003) C-peptide compared with T-cell− patients. These data suggest that measuring T-cell responses to multiple islet proteins in phenotypic type 2 diabetic patients improves identification of patients with autoimmune diabetes and delineates those who have a more severe β-cell lesion compared with Ab assessment alone.
Loss-of-Function Mutation in Toll-Like Receptor 4 Prevents Diet-Induced Obesity and Insulin Resistance
Authors: Tsukumo Daniela M.L. , Carvalho-Filho Marco A. , Carvalheira José B.C. , Prada Patrícia O. , Hirabara Sandro M. , Schenka André A. , Araújo Eliana P. , Vassallo José , Curi Rui , Velloso Lício A. , Saad Mario J.A.
Published: July 2007

Obesity is associated with insulin resistance and a state of abnormal inflammatory response. The Toll-like receptor (TLR)4 has an important role in inflammation and immunity, and its expression has been reported in most tissues of the body, including the insulin-sensitive ones. Because it is activated by lipopolysaccharide and saturated fatty acids, which are inducers of insulin resistance, TLR4 may be a candidate for participation in the cross-talk between inflammatory and metabolic signals. Here, we show that C3H/HeJ mice, which have a loss-of-function mutation in TLR4, are protected against the development of diet-induced obesity. In addition, these mice demonstrate decreased adiposity, increased oxygen consumption, a decreased respiratory exchange ratio, improved insulin sensitivity, and enhanced insulin-signaling capacity in adipose tissue, muscle, and liver compared with control mice during high-fat feeding. Moreover, in these tissues, control mice fed a high-fat diet show an increase in IκB kinase complex and c-Jun NH2-terminal kinase activity, which is prevented in C3H/HeJ mice. In isolated muscles from C3H/HeJ mice, protection from saturated fatty acid–induced insulin resistance is observed. Thus, TLR4 appears to be an important mediator of obesity and insulin resistance and a potential target for the therapy of these highly prevalent medical conditions.
5-Aminoimidazole-4-Carboxamide 1-β-d-Ribofuranoside Acutely Stimulates Skeletal Muscle 2-Deoxyglucose Uptake in Healthy Men
Authors: Cuthbertson Daniel J. , Babraj John A. , Mustard Kirsteen J.W. , Towler Mhairi C. , Green Kevin A. , Wackerhage Henning , Leese Graeme P. , Baar Keith , Thomason-Hughes Michaela , Sutherland Calum , Hardie D. Grahame , Rennie Michael J.
Published: July 2007

Activation of AMP-activated protein kinase (AMPK) in rodent muscle by exercise, metformin, 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR), and adiponectin increases glucose uptake. The aim of this study was to determine whether AICAR stimulates muscle glucose uptake in humans. We studied 29 healthy men (aged 26 ± 8 years, BMI 25 ± 4 kg/m2 [mean ± SD]). Rates of muscle 2-deoxyglucose (2DG) uptake were determined by measuring accumulation of total muscle 2DG (2DG and 2DG-6-phosphate) during a primed, continuous 2DG infusion. The effects of AICAR and exercise on muscle AMPK activity/phosphorylation and 2DG uptake were determined. Whole-body glucose disposal was compared before and during AICAR with the euglycemic-hyperinsulinemic clamp. Muscle 2DG uptake was linear over 9 h (R2 = 0.88 ± 0.09). After 3 h, 2DG uptake increased 2.1 ± 0.8- and 4.7 ± 1.7-fold in response to AICAR or bicycle exercise, respectively. AMPK α1 and α2 activity or AMPK phosphorylation was unchanged after 20 min or 3 h of AICAR, but AMPK phosphorylation significantly increased immediately and 3 h after bicycle exercise. AICAR significantly increased phosphorylation of extracellular signal–regulated kinase 1/2, but phosphorylation of β-acetyl-CoA carboxylase, glycogen synthase, and protein kinase B or insulin receptor substrate-1 level was unchanged. Mean whole-body glucose disposal increased by 7% with AICAR from 9.3 ± 0.6 to 10 ± 0.6 mg · kg−1 · min−1 (P < 0.05). In healthy people, AICAR acutely stimulates muscle 2DG uptake with a minor effect on whole-body glucose disposal.
Podocyte Detachment and Reduced Glomerular Capillary Endothelial Fenestration in Human Type 1 Diabetic Nephropathy
Authors: Toyoda Masao , Najafian Behzad , Kim Youngki , Caramori M. Luiza , Mauer Michael
Published: July 2007

The aim of this study was to investigate the structural characteristics of podocytes and endothelial cells in diabetic nephropathy. We studied 18 patients with type 1 diabetes (seven normoalbuminuric, six microalbuminuric, and five proteinuric), and six normal control subjects. Groups were not different for age. Type 1 diabetic groups were not different for diabetes duration or age at diabetes onset. Podocyte foot process width (FPW), fraction of glomerular basement membrane (GBM) surface with intact nondetached foot processes (IFP), fraction of glomerular capillary luminal surface covered by fenestrated endothelium [SS(Fenestrated/cap)] and classic diabetic glomerulopathy lesions were morphometrically measured. Albumin excretion (AER) and glomerular filtration (GFR) rates were also measured. GFR correlated inversely and AER directly with GBM and mesangial measurements in diabetic patients. FPW correlated inversely with GFR (r = −0.71, P = 0.001) and directly with AER (r = 0.66, P = 0.003), GBM, and mesangial parameters. The GBM fraction covered by IFP was decreased in proteinuric versus control subjects (P = 0.001), normoalbuminuric patients (P = 0.0002) and microalbuminuric patients (P = 0.04) and correlated with renal structural and functional parameters, including AER (r = −0.52, P = 0.03). Only 78% of GBM was covered by IFP in proteinuric patients. SS(Fenestrated/cap) was reduced in normoalbuminuric (P = 0.03), microalbuminuric (P = 0.03), and proteinuric (P = 0.002) patients versus control subjects. SS(Fenestrated/cap) correlated with mesangial fractional volume per glomerulus (r = −0.57, P = 0.01), IFP (r = 0.61, P = 0.007), and FPW (r = −0.58, P = 0.01). These novel studies document that podocyte detachment and diminished endothelial cell fenestration are related to classical diabetic nephropathy lesions and renal function in type 1 diabetic patients and support a need for further studies of podocyte/GBM adherence and podocyte/endothelial cell functional interactions in diabetic nephropathy.
Multiple Variants in Vascular Endothelial Growth Factor (VEGFA) Are Risk Factors for Time to Severe Retinopathy in Type 1 Diabetes
Authors: Al-Kateb Hussam , Mirea Lucia , Xie Xinlei , Sun Lei , Liu Michelle , Chen Hongtao , Bull Shelley B. , Boright Andrew P. , Paterson Andrew D. ,   for the DCCT/EDIC Research Group
Published: July 2007

OBJECTIVE—We sought to determine if any common variants in the gene for vascular endothelial growth factor (VEGFA) are associated with long-term renal and retinal complications in type 1 diabetes.

RESEARCH DESIGN AND METHODS—A total of 1,369 Caucasian subjects with type 1 diabetes from the Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Interventions and Complications (EDIC) Study had an average of 17 retinal photographs and 10 renal measures over 15 years. In the DCCT/EDIC, we studied 18 single nucleotide polymorphisms (SNPs) in VEGFA that represent all linkage disequilibrium bins (pairwise r2 ≥ 0.64) and tested them for association with time to development of severe retinopathy, three or more step progression of retinopathy, clinically significant macular edema, persistent microalbuminuria, and severe nephropathy.

RESULTS—In a global multi-SNP test, there was a highly significant association of VEGFA SNPs with severe retinopathy (P = 6.8 × 10−5)—the four other outcomes were all nonsignificant. In survival analyses controlling for covariate risk factors, eight SNPs showed significant association with severe retinopathy (P < 0.05). The most significant single SNP association was rs3025021 (hazard ratio 1.37 [95% CI 1.13–1.66], P = 0.0017). Family-based analyses of severe retinopathy provide evidence of excess transmission of C at rs699947 (P = 0.029), T at rs3025021 (P = 0.013), and the C-T haplotype from both SNPs (P = 0.035). Multi-SNP regression analysis including 15 SNPs, and allowing for pairwise interactions, independently selected 6 significant SNPs (P < 0.05).

CONCLUSIONS—These data demonstrate that multiple VEGFA variants are associated with the development of severe retinopathy in type 1 diabetes.
Effect of ageing on optical and thermal properties of YBa2Cu3O7–δ
Authors: Nikolic Pantelija M. , Vujatovic Stevan S. , Paraskevopoulos Konstantinos M. , Zorbas Kostas T. , Savic Slavica M. , Nikolic Maria Vesna , Aleksic Obrad S. , Ristic Momcilo M.
Published: July 2007

Photoacoustic phase and amplitude spectra of YBa2Cu3O7 – δ samples synthesised 18 years ago were measured and numerically analysed. The value of thermal diffusivity obtained on the aged samples was lower than the values obtained for fresh samples. Far infrared spectra of YBa2Cu3O7 – δ samples synthesised 18 years ago were recorded and analysed enabling determination of reflectivity peak phonon frequencies present at room temperature. X-ray diffraction analysis and far infrared reflectivity measurements enabled determination of a slight change of the oxygen content with time. The YBa2Cu3O7 – δ samples synthesised 18 years ago still remained superconducting, which was confirmed by resistivity measurements of the samples between 50 K and room temperature where the critical temperature was reduced from 93 to 73 K. Atomic force microscopy, scanning electron microscopy and energy dispersive X-ray microanalytical measurements were also performed confirming the composition of the sample.