Differentiation of mitochondrial DNA and Y chromosomes in Russian populations.
The genetic composition of the Russian population was investigated by analyzing both mitochondrial DNA (mtDNA) and Y-chromosome loci polymorphisms that allow for the different components of a population gene pool to be studied, depending on the mode of DNA marker inheritance. mtDNA sequence variation was examined by using hypervariable segment I (HVSI) sequencing and restriction analysis of the haplogroup-specific sites in 325 individuals representing 5 Russian populations from the European part of Russia. The Y-chromosome variation was investigated in 338 individuals from 8 Russian populations (including 5 populations analyzed for mtDNA variation) using 12 binary markers. For both uniparental systems most of the observed haplogroups fell into major West Eurasian haplogroups (97.9% and 99.7% for mtDNA and Y-chromosome haplogroups, respectively). Multidimensional scaling analysis based on pairwise F(ST) values between mtDNA HVSI sequences in Russians compared to other European populations revealed a considerable heterogeneity of Russian populations; populations from the southern and western parts of Russia are separated from eastern and northern populations. Meanwhile, the multidimensional scaling analysis based on Y-chromosome haplogroup F(ST) values demonstrates that the Russian gene pool is close to central-eastern European populations, with a much higher similarity to the Baltic and Finno-Ugric male pools from northern European Russia. This discrepancy in the depth of penetration of mtDNA and Y-chromosome lineages characteristic for the most southwestern Russian populations into the east and north of eastern Europe appears to indicate that Russian colonization of the northeastern territories might have been accomplished mainly by males rather than by females.


Efficiency of E2-p7 processing modulates production of infectious hepatitis C virus.
Previous studies indicate that the processing of hepatitis C virus (HCV) E2-p7-NS2 precursor mediated by host signal peptidase is relatively inefficient, resulting in the accumulation of E2-p7-NS2 and E2-p7 precursors in addition to E2 in mammalian cells. In this study, we discovered that a significant inhibition of the processing at an E2-p7 junction site is detrimental for HCV production, whether it was caused by the mutations in p7 or by the strategic introduction of a mutation at a terminal residue of E2 to block the signal peptidase-mediated cleavage of this junction site. However, complete separation of E2 and p7 by inserting an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) between these two proteins also moderately inhibited virus production. These results indicate that optimal processing of the E2-p7 junction site is critical for efficient HCV production. We further demonstrated that disrupting E2-p7 processing inhibits both NS2 localization to the putative virus assembly sites near lipid droplets (LD) and NS2 interaction with NS3 and E2. However, the impact, if any, of the p7-NS2 processing efficiency on HCV production seems relatively minor. In conclusion, these results imply that effective release of E2 and p7 from the precursor E2-p7 promotes HCV production by enhancing NS2-associated virus assembly complex formation near LD.


Harmine enhances type H vessel formation and prevents bone loss in ovariectomized mice.
Recently, researchers identified a distinct vessel subtype called type H vessels that couple angiogenesis and osteogenesis. We previously found that type H vessels are reduced in ovariectomy (OVX)-induced osteoporotic mice, and preosteoclasts are able to secrete platelet-derived growth factor-BB (PDGF-BB) to stimulate type H vessel formation and thereby to promote osteogenesis. This study aimed to explore whether harmine, a β-carboline alkaloid, is capable of preventing bone loss in OVX mice by promoting preosteoclast PDGF-BB-induced type H vessel formation. The impact of harmine on osteoclastogenesis of RANKL-stimulated RAW264.7 cells was verified by gene expression analysis and tartrate-resistant acid phosphatase (TRAP) staining. Enzyme-linked immunosorbent assay (ELISA) was conducted to test PDGF-BB production by preosteoclasts. A series of angiogenesis-related assays in vitro were performed to assess the pro-angiogenic effects of the conditioned media from RANKL-stimulated RAW264.7 cells treated with or without harmine. Meanwhile, the role of PDGF-BB in this process was determined. In vivo, OVX mice were intragastrically administrated with harmine emulsion or an equal volume of vehicle. 2 months later, bone samples were collected for µCT, histological, immunohistochemical and immunofluorescent analyses to evaluate bone mass, osteogenic and osteoclastic activities, as well as the numbers of type H vessels. Bone marrow PDGF-BB concentrations were assessed by ELISA. Exposure of RANKL-stimulated RAW264.7 cells to harmine enhanced the formation of preosteoclasts and the production of PDGF-BB. Harmine augmented the ability of RANKL-stimulated RAW264.7 cells to promote angiogenesis of endothelial cells, whereas the effect was blocked by PDGF-BB inhibition. In vivo, the oral administration of harmine emulsion to OVX mice resulted in enhanced trabecular bone mass and osteogenic responses, increased numbers of preosteoclasts, as well as reduced numbers of osteoclasts and fat cells. Moreover, OVX mice treated with harmine exhibited higher levels of bone marrow PDGF-BB and much more type H vessels in bone. Harmine may exert bone-sparing effects by suppression of osteoclast formation and promotion of preosteoclast PDGF-BB-induced angiogenesis.


Melatonin Attenuates Pulmonary Hypertension in Chronically Hypoxic Rats.
Chronic hypoxia induces pulmonary hypertension and vascular remodeling, which are clinically relevant to patients with chronic obstructive pulmonary disease (COPD) associated with a decreased level of nitric oxide (NO). Oxidative stress and inflammation play important roles in the pathophysiological processes in COPD. We examined the hypothesis that daily administration of melatonin (10 mg/kg) mitigates the pulmonary hypertension and vascular remodeling in chronically hypoxic rats. The right ventricular systolic pressure (RVSP) and the thickness of pulmonary arteriolar wall were measured from normoxic control, vehicle- and melatonin-treated hypoxic rats exposed to 10% O₂ for 14 days. Levels of markers for oxidative stress (malondialdhyde) and inflammation (tumor necrosis factor-α (TNFα), inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2)) and the expressions of total endothelial NO synthase (eNOS) and phosphorylated eNOS at serine1177 (ser1177) were determined in the lung tissue. We found that the RVSP and the thickness of the arteriolar wall were significantly increased in the vehicle-treated hypoxic animals with elevated levels of malondialdhyde and mRNA expressions of the inflammatory mediators, when compared with the normoxic control. In addition, the phosphorylated eNOS (ser1177) level was significantly decreased, despite an increased eNOS expression in the vehicle-treated hypoxic group. Melatonin treatment significantly attenuated the levels of RVSP, thickness of the arteriolar wall, oxidative and inflammatory markers in the hypoxic animals with a marked increase in the eNOS phosphorylation in the lung. These results suggest that melatonin attenuates pulmonary hypertension by antagonizing the oxidative injury and restoration of NO production.


REDUCING CSF PARTIAL VOLUME EFFECTS TO ENHANCE DIFFUSION TENSOR IMAGING METRICS OF BRAIN MICROSTRUCTURE.
Technological advances over recent decades now allow for in vivo observation of human brain tissue through the use of neuroimaging methods. While this field originated with techniques capable of capturing macrostructural details of brain anatomy, modern methods such as diffusion tensor imaging (DTI) that are now regularly implemented in research protocols have the ability to characterize brain microstructure. DTI has been used to reveal subtle micro-anatomical abnormalities in the prodromal phase ofº various diseases and also to delineate "normal" age-related changes in brain tissue across the lifespan. Nevertheless, imaging artifact in DTI remains a significant limitation for identifying true neural signatures of disease and brain-behavior relationships. Cerebrospinal fluid (CSF) contamination of brain voxels is a main source of error on DTI scans that causes partial volume effects and reduces the accuracy of tissue characterization. Several methods have been proposed to correct for CSF artifact though many of these methods introduce new limitations that may preclude certain applications. The purpose of this review is to discuss the complexity of signal acquisition as it relates to CSF artifact on DTI scans and review methods of CSF suppression in DTI. We will then discuss a technique that has been recently shown to effectively suppress the CSF signal in DTI data, resulting in fewer errors and improved measurement of brain tissue. This approach and related techniques have the potential to significantly improve our understanding of "normal" brain aging and neuropsychiatric and neurodegenerative diseases. Considerations for next-level applications are discussed.


Effects of chronic administration of nicotine on prolactin release in the rat: inactivation of prolactin response by repeated injections of nicotine.
The effects of chronic injections of nicotine on nicotine-induced prolactin release in the rat were measured and compared to the effects of this treatment on [3H]acetylcholine binding to nicotinic cholinergic sites in the hypothalamus. Treatment with nicotine for 10 days (s.c. injections twice daily) abolished prolactin release in response to an acute i.v. injection of nicotine given 2, 6 or 8 days after the last of the chronic injections of nicotine. At each of these time points, the binding of [3H]acetylcholine in the hypothalamus from rats treated chronically with nicotine was significantly higher than in the hypothalamus from control rats. By 14 days after the last chronic injection of nicotine, the prolactin response to an acute injection of nicotine was restored. Coinciding with the return of the nicotine-induced prolactin response, the binding of [3H]acetylcholine had returned to control values. These results are consistent with the hypothesis that nicotine inactivates nicotinic cholinergic receptors in brain by an allosteric mechanism, and that prolonged inactivation of nicotinic cholinergic receptors leads to their increased number.


The binding of monomeric C-reactive protein (mCRP) to Integrins αvβ3 and α4β1 is related to its pro-inflammatory action.
The prototypic acute phase reactant C-reactive protein (CRP) is not only a marker but also a potential contributor to inflammatory diseases. CRP exists as the circulating native, pentameric CRP (pCRP) and the monomeric isoform (mCRP), formed as a result of a dissociation process of pCRP. mCRP is highly pro-inflammatory, but pCRP is not. The mechanism of pro-inflammatory action of mCRP is unclear. We studied the role of integrins in pro-inflammatory action of mCRP. Docking simulation of interaction between mCRP and integrin αvβ3 predicted that mCRP binds to αvβ3 well. We found that mCRP actually bound to integrins αvβ3 and α4β1 well. Antagonists to αvβ3 or α4β1 effectively suppressed the interaction, suggesting that the interaction is specific. Using an integrin β1 mutant (β1-3-1) that has a small fragment from the ligand binding site of β3, we showed that mCRP bound to the classical RGD-binding site in αvβ3. We studied the role of integrins in CRP signaling in monocytic U937 cells. Integrins αvβ3 and α4β1 specifically mediated binding of mCRP to U937 cells. mCRP induced AKT phosphorylation, but not ERK1/2 phosphorylation, in U937 cells. Notably, mCRP induced robust chemotaxis in U937 cells, and antagonists to integrins αvβ3 and α4β1 and an inhibitor to phosphatidylinositide 3-kinase, but not an MEK inhibitor, effectively suppressed mCRP-induced chemotaxis in U937 cells. These results suggest that the integrin and AKT/phosphatidylinositide 3-kinase pathways play a role in pro-inflammatory action of mCRP in U937 cells. In contrast, pCRP is predicted to have a limited access to αvβ3 due to steric hindrance in the simulation. Consistent with the prediction, pCRP was much less effective in integrin binding, chemotaxis, or AKT phosphorylation. These findings suggest that the ability of CRP isoforms to bind to the integrins is related to their pro-inflammatory action.


Hollow polycaprolactone composite fibers for controlled magnetic responsive antifungal drug release.
Hollow magnetic fibers for trigger based drug release were synthesized using one-step co-axial electrospinning (COX-ES). This was achieved by encapsulating the antifungal active 'ketoconazole' (KCZ) and iron oxide (Fe3O4) nanoparticles (NPs) in composite form within the core shell polymeric matrix material (polycaprolactone, PCL) during the COX-ES process. Dimethyl silicone oil was used as the inner core (liquid) of co-flowing solutions, which subsequently perfused out of the two-phase electrospun microstructures to form hollow fibers. Resulting drug-loaded magnetic hollow fibers were characterized using optical microscopy, scanning electron microscopy and Fourier Transform Infra-Red. The tensile strength and magnetization properties of composite fibers were also assessed. KCZ drug concentration in electrospinning solutions strongly influenced resulting fiber morphology, drug loading efficiency and release. Expedited drug release during a slow-sustained phase was demonstrated through the application of an auxiliary magnetic field. Variations in tensile strength (∼1.3-6.3MPa) were due to composite fiber components compromising polymer chain integrity. In-vitro cell studies (using human cervical carcinoma cell lines) demonstrated fiber biocompatibility. The present study demonstrates the potential application of magnetic hollow fibers for controlled treatment of fungal infections and antimicrobial indications.


Focal adhesion kinase is a load-dependent governor of the slow contractile and oxidative muscle phenotype.
Striated muscle exhibits a pronounced structural-functional plasticity in response to chronic alterations in loading. We assessed the implication of focal adhesion kinase (FAK) signalling in mechano-regulated differentiation of slow-oxidative muscle. Load-dependent consequences of FAK signal modulation were identified using a multi-level approach after electrotransfer of rat soleus muscle with FAK-expression plasmid vs. empty plasmid-transfected contralateral controls. Muscle fibre-targeted over-expression of FAK in anti-gravitational muscle for 9 days up-regulated transcript levels of gene ontologies underpinning mitochondrial metabolism and contraction in the transfected belly portion. Concomitantly, mRNA expression of the major fast-type myosin heavy chain (MHC) isoform, MHC2A, was reduced. The promotion of the slow-oxidative expression programme by FAK was abolished after co-expression of the FAK inhibitor FAK-related non-kinase (FRNK). Elevated protein content of MHC1 (+9%) and proteins of mitochondrial respiration (+165-610%) with FAK overexpression demonstrated the translation of transcript differentiation in targeted muscle fibres towards a slow-oxidative muscle phenotype. Coincidentally MHC2A protein was reduced by 50% due to protection of muscle from de-differentiation with electrotransfer. Fibre cross section in FAK-transfected muscle was elevated by 6%. The FAK-modulated muscle transcriptome was load-dependent and regulated in correspondence to tyrosine 397 phosphorylation of FAK. In the context of overload, the FAK-induced gene expression became manifest at the level of contraction by a slow transformation and the re-establishment of normal muscle force from the lowered levels with transfection. These results highlight the analytic power of a systematic somatic transgene approach by mapping a role of FAK in the dominant mechano-regulation of muscular motor performance via control of gene expression.


Inducible NO synthase confers chemoresistance in head and neck cancer by modulating survivin.
The dual role of the inducible NO synthase (iNOS) and NO signaling in head and neck squamous cell carcinoma (HNSCC) is a complex and can both promote or inhibit tumor progression. However, the underlying molecular mechanisms are not yet resolved in detail. We show for the first time that conditions, favoring low NO levels conferred resistance against cisplatin/taxol-induced apoptosis in HNSCC cell lines. Cytoprotection was mediated by survivin, because we observed its upregulation subsequent to low doses of the NO donors S-nitroso-N-acetyl-penicillamine (SNAP) and sodium nitroprusside (SNP) or ectopic expression of physiologic amounts of iNOS. Also, RNAi-mediated depletion of survivin blocked NOs anti-apoptotic effects. Induction of survivin involves activation of the phosphatidylinositol-3-kinase/Akt (PI3K/Akt) pathway, which was antagonized by the PI3K-inhibitor LY294002. Importantly, application of the iNOS-specific inhibitor 1400W combined with RNAi-mediated downregulation of survivin cooperatively enhanced drug-induced cell death. The iNOS/survivin-axis appears to be also of clinical relevance since immunohistochemistry revealed that iNOS expression correlated with enhanced survivin levels in HNSCC specimens. In contrast, high NO concentrations suppressed survivin levels in HNSCC but also in non-malignant cells resulting in apoptosis. Cell death induced by high amounts of SNAP/SNP or by strong overexpression of iNOS involved activation of p38MAP-kinase, which was counteracted by the p38MAP-kinase inhibitor SB202190. Here, we provide evidence for a novel molecular mechanism how NO signaling may contribute to therapy resistance in HNSCC by modulating survivin expression. Our data further suggest pursuing pharmacogenetic iNOS/survivin-targeting approaches as potential therapeutic strategies in head and neck cancer.


Time course of changes in hepatic metabolism in response to sepsis in the rat: impairment of gluconeogenesis and ketogenesis in vitro.
The time course (12, 24 and 48 h) of changes in blood metabolites, and in gluconeogenesis and ketogenesis, in isolated hepatocytes from rats made septic by caecal ligation and puncture was measured. Blood glucose was not significantly different in septic rats, but lactate was increased at 12, 24 and 48 h; pyruvate and alanine were increased at 48 h. The blood ketone body concentrations were decreased at all times studied after induction of sepsis. These changes were accompanied by increased plasma insulin in the septic rats. The rate of hepatic lipogenesis in vivo was increased at 24 and 48 h. There were appreciable increases in the hepatic concentrations of alanine (200%), lactate (200%) and pyruvate (100%) as well as other intermediates in the gluconeogenic pathway. The hepatic concentrations of acetyl-CoA and ketone bodies were decreased. The rate of gluconeogenesis from added lactate, pyruvate, alanine and glutamine was depressed in isolated hepatocytes from septic rats at 24 and 48 h. The basal rate of ketogenesis or the rate from butyrate in isolated hepatocytes was not significantly altered by sepsis, whereas the rate from oleate was decreased at all time points. It is concluded that there is an impairment of the capacity for gluconeogenesis and ketogenesis in livers of septic rats. The latter may be due to decreased entry of long-chain acyl-CoA into the mitochondria for oxidation. The possibility that these changes are in part brought about by the hyperinsulinaemia associated with the sepsis is discussed.


[The great virus comeback].
Viruses have been considered for a long time as by-products of biological evolution. This view is changing now as a result of several recent discoveries. Viral ecologists have shown that viral particles are the most abundant biological entities on our planet, whereas metagenomic analyses have revealed an unexpected abundance and diversity of viral genes in the biosphere. Comparative genomics have highlighted the uniqueness of viral sequences, in contradiction with the traditional view of viruses as pickpockets of cellular genes. On the contrary, cellular genomes, especially eukaryotic ones, turned out to be full of genes derived from viruses or related elements (plasmids, transposons, retroelements and so on). The discovery of unusual viruses infecting archaea has shown that the viral world is much more diverse than previously thought, ruining the traditional dichotomy between bacteriophages and viruses. Finally, the discovery of giant viruses has blurred the traditional image of viruses as small entities. Furthermore, essential clues on virus history have been obtained in the last ten years. In particular, structural analyses of capsid proteins have uncovered deeply rooted homologies between viruses infecting different cellular domains, suggesting that viruses originated before the last universal common ancestor (LUCA). These studies have shown that several lineages of viruses originated independently, i.e., viruses are polyphyletic. From the time of LUCA, viruses have coevolved with their hosts, and viral lineages can be viewed as lianas wrapping around the trunk, branches and leaves of the tree of life. Although viruses are very diverse, with genomes encoding from one to more than one thousand proteins, they can all be simply defined as organisms producing virions. Virions themselves can be defined as infectious particles made of at least one protein associated with the viral nucleic acid, endowed with the capability to protect the viral genome and ensure its delivery to the infected cell. These definitions, which clearly distinguish viruses from plasmids, suggest that infectious RNA molecules that only encode an RNA replicase presently classified among viruses by the ICTV (International Committee for the Taxonomy of Viruses) into families of Endornaviridae and Hypoviridae are in fact RNA plasmids. Since a viral genome should encode for at least one structural protein, these definitions also imply that viruses originated after the emergence of the ribosome in an RNA-protein cellular world. Although virions are the hallmarks of viruses, viruses and virions should not be confused. The infection transforms the ribocell (cell encoding ribosomes and dividing by binary fission) into a virocell (cell producing virions) or ribovirocell (cell that produces virions but can still divide by binary fission). In the ribovirocell, two different organisms, defined by their distinct evolutionary histories, coexist in symbiosis in the same cell. The virocells or ribovirocells are the living forms of the virus, which can be in fine considered to be a living organism. In the virocell, the metabolism is reorganized for the production of virions, while the ability to capture and store free energy is retained, as in other cellular organisms. In the virocell, viral genomes replicate, recombine and evolve, leading to the emergence of new viral proteins and potentially novel functions. Some of these new functions can be later on transferred to the cell, explaining how viruses can play a major (often underestimated) role in the evolution of cellular organisms. The virocell concept thus helps to understand recent hypotheses suggesting that viruses played a critical role in major evolutionary transitions, such as the origin of DNA genomes or else the origin of the eukaryotic nucleus. Finally, it is more and more recognized that viruses are the major source of variation and selection in living organisms (both viruses and cells), the two pillars of darwinism. One can thus conclude that the continuous interaction between viruses and cells, all along the history of life, has been, and still is, a major engine of biological evolution.


Therapeutic options for the treatment of tinea capitis caused by Trichophyton species: griseofulvin versus the new oral antifungal agents, terbinafine, itraconazole, and fluconazole.
Tinea capitis is a relatively common fungal infection of childhood. Griseofulvin has been the mainstay of management. However, newer oral antifungal agents are being used more frequently. A multicenter, prospective, randomized, single-blinded, non-industry-sponsored study was conducted in centers in Canada and South Africa to determine the relative efficacy and safety of griseofulvin, terbinafine, itraconazole, and fluconazole in the treatment of tinea capitis caused by Trichophyton species. The regimens for treating tinea capitis were griseofulvin microsize 20 mg/kg/day x 6 weeks, terbinafine [> 40 kg, one 250 mg tablet; 20-40 kg, 125 mg (half of a 250 mg tablet); < 20 kg, 62.5 mg (one-quarter of a 250 mg tablet)] x 2-3 weeks, itraconazole 5 mg/kg/day x 2-3 weeks, and fluconazole 6 mg/kg/day x 2-3 weeks. Patients were asked to return at weeks 4, 8, and 12 from the start of the study. Griseofulvin was administered for 6 weeks and the final evaluation was at week 12. Terbinafine, itraconazole, and fluconazole were administered for 2 weeks and the patient evaluated 4 weeks from the start of therapy. At this time, if clinically indicated, one extra week of therapy was given. There were 200 patients randomized to four treatment groups (50 in each group). At the final evaluation at week 12, the number of evaluable patients were griseofulvin, 46; terbinafine, 48; itraconazole, 46; and fluconazole, 46. Patients who discontinued therapy or were lost to follow-up were griseofulvin, 1/3; itraconazole, 0/4; terbinafine, 0/4; and fluconazole, 0/4. The causative organisms were Trichophyton tonsurans and T. violaceum species. Patients were regarded as effectively treated at week 12 if there was mycologic cure and either clinical cure or only a few residual symptoms. Effective treatment was recorded in, intention to treat, griseofulvin (46 of 50, 92.0%), terbinafine (47 of 50, 94.0%), itraconazole (43 of 50, 86.0%), and fluconazole (42 of 50, 84.0%) (p=0.33). Adverse effects were reported only in the griseofulvin group (gastrointestinal effects in six patients). Discontinuation from therapy due to adverse effects occurred only in the griseofulvin group (nausea in one patient). For the treatment of tinea capitis caused by the Trichophyton species, in this study, griseofulvin given for 6 weeks is similar in efficacy to terbinafine, itraconazole, and fluconazole given for 2-3 weeks. Each of the agents has a favorable adverse-effects profile.


Dissemination of clinical isolates of Klebsiella oxytoca harboring CMY-31, VIM-1, and a New OXY-2-type variant in the community.
The aim of the present study was to investigate the epidemiological link of multidrug-resistant Klebsiella oxytoca isolates causing community-onset infections among patients attending our outpatient department and to investigate the underlying resistance mechanisms. The isolates were tested by agar dilution MICs, phenotypic carbapenemase testing, enterobacterial repetitive intergenic consensus-PCR, and pulsed-field gel electrophoresis (PFGE). PCR assays and nucleotide sequencing were employed for the identification of bla gene types and the mapping of the integron-containing metallo-β-lactamase (MBL) gene. During the study period (January 2005 to April 2007), nine broad-spectrum cephalosporin-resistant K. oxytoca clinical isolates were prospectively collected from separate outpatients with urinary tract infections. In all cases, the patients had been hospitalized or exposed to health care facilities during the preceding year. Molecular typing revealed that all isolates belonged to the same K. oxytoca clonal type, which contained five PFGE subtypes. A novel chromosomal OXY-2 β-lactamase type variant (OXY-2-9) was detected in all isolates, but no mutations in the promoter region justifying bla(OXY) gene overproduction were detected. In addition, all isolates harbored the plasmidic CMY-31 (LAT-4) AmpC cephalosporinase, while three of them harbored VIM-1 MBL in a class 1 integron structure. This is the first study to present the dissemination in the community of multidrug-resistant K. oxytoca isolates causing extrahospital infections.


Local recurrence and assessment of sentinel lymph node biopsy in deep soft tissue leiomyosarcoma of the extremities.
Leiomyosarcoma of deep soft tissues of the extremities is a rare malignant tumour treated primarily by surgery. The incidence of local recurrence and lymph node metastasis is uncertain and it is not known whether a sentinel lymph node biopsy is indicated in these tumours. A retrospective review of patients treated for extremity deep soft tissue leiomyosarcoma at our institution over a 10-year period was conducted. Patients developing local recurrence or lymph node metastasis were identified. The presence or absence of lymphatics in the primary tumours was assessed by immunohistochemical expression of LYVE-1 and podoplanin. 27 patients (mean age 62 years) were included in the study. 15 were female and 12 male. Lymph node metastasis was seen in only two cases (7%); intratumoural lymphatics were identified in the primary tumours of both these cases. Local recurrence occurred in 25.9% of cases despite complete excision and post-operative radiotherapy; the mean time to recurrence was 10.1 months. On the basis of this study, we do not advocate sentinel lymph node biopsy in this group of patients except in those cases in which intratumoural lymphatics can be demonstrated. Close follow up is important especially for high grade leiomyosarcomas, particularly in the first year, as these tumours have a high incidence of local recurrence.


Visualizing the orientational dependence of an intermolecular potential.
Scanning probe microscopy can now be used to map the properties of single molecules with intramolecular precision by functionalization of the apex of the scanning probe tip with a single atom or molecule. Here we report on the mapping of the three-dimensional potential between fullerene (C60) molecules in different relative orientations, with sub-Angstrom resolution, using dynamic force microscopy (DFM). We introduce a visualization method which is capable of directly imaging the variation in equilibrium binding energy of different molecular orientations. We model the interaction using both a simple approach based around analytical Lennard-Jones potentials, and with dispersion-force-corrected density functional theory (DFT), and show that the positional variation in the binding energy between the molecules is dominated by the onset of repulsive interactions. Our modelling suggests that variations in the dispersion interaction are masked by repulsive interactions even at displacements significantly larger than the equilibrium intermolecular separation.


Molecular Impact of Electronic Cigarette Aerosol Exposure in Human Bronchial Epithelium.
Little evidence is available regarding the physiological effects of exposure to electronic cigarette (ECIG) aerosol. We sought to determine the molecular impact of ECIG aerosol exposure in human bronchial epithelial cells (HBECs). Gene-expression profiling was conducted in primary grown at air liquid interface and exposed to 1 of 4 different ECIG aerosols, traditional tobacco cigarette (TCIG) smoke, or clean air. Findings were validated experimentally with quantitative polymerase chain reaction and a reactive oxygen species immunoassay. Using gene set enrichment analysis, signatures of in vitro ECIG exposure were compared with those generated from bronchial epithelial brushings of current TCIG smokers and former TCIG smokers currently using ECIGs. We found 546 genes differentially expressed across the ECIG, TCIG, and air-exposed groups of HBECs (ANOVA; FDR q < .05; fold change > 1.5). A subset of these changes were shared between TCIG- and ECIG-exposed HBECs. ECIG exposure induced genes involved in oxidative and xenobiotic stress pathways and increased a marker of reactive oxygen species production in a dose-dependent manner. ECIG exposure decreased expression of genes involved in cilia assembly and movement. Furthermore, gene-expression differences observed in vitro were concordant with differences observed in airway epithelium collected from ECIG users (q < .01). In summary, our data suggest that ECIG aerosol can induce gene-expression changes in bronchial airway epithelium in vitro, some of which are shared with TCIG smoke. These changes were generally less pronounced than the effects of TCIG exposure and were more pronounced in ECIG products containing nicotine than those without nicotine. Our data further suggest that the gene-expression alterations seen with the in vitro exposure system reflects the physiological effects experienced in vivo by ECIG users.


CD13 is a therapeutic target in human liver cancer stem cells.
Cancer stem cells (CSCs) are generally dormant or slowly cycling tumor cells that have the ability to reconstitute tumors. They are thought to be involved in tumor resistance to chemo/radiation therapy and tumor relapse and progression. However, neither their existence nor their identity within many cancers has been well defined. Here, we have demonstrated that CD13 is a marker for semiquiescent CSCs in human liver cancer cell lines and clinical samples and that targeting these cells might provide a way to treat this disease. CD13+ cells predominated in the G0 phase of the cell cycle and typically formed cellular clusters in cancer foci. Following treatment, these cells survived and were enriched along the fibrous capsule where liver cancers usually relapse. Mechanistically, CD13 reduced ROS-induced DNA damage after genotoxic chemo/radiation stress and protected cells from apoptosis. In mouse xenograft models, combination of a CD13 inhibitor and the genotoxic chemotherapeutic fluorouracil (5-FU) drastically reduced tumor volume compared with either agent alone. 5-FU inhibited CD90+ proliferating CSCs, some of which produce CD13+ semiquiescent CSCs, while CD13 inhibition suppressed the self-renewing and tumor-initiating ability of dormant CSCs. Therefore, combining a CD13 inhibitor with a ROS-inducing chemo/radiation therapy may improve the treatment of liver cancer.


Early treatment of acute submacular haemorrhage secondary to wet AMD using intravitreal tissue plasminogen activator, C3F8, and an anti-VEGF agent.
PurposeAcute submacular haemorrhage secondary to wet age-related macular degeneration (AMD) has a poor prognosis for which there is currently no 'gold standard' treatment. We evaluated the efficacy of early treatment using intravitreal triple therapy of tissue plasminogen activator (tPA), expansile gas, and an anti-VEGF agent.MethodsThis retrospective case series included eight patients presenting with acute submacular haemorrhage involving the fovea. All patients received treatment with 50 μg (0.05 ml) tPA, 0.3 ml 100% perfluoropropane (C3F8), and an anti-VEGF agent (0.05 mg Ranibizumab or 1.25 mg Bevacizumab in 0.05 ml) administered via intravitreal injection. An anterior chamber paracentesis post injection or vitreous tap was performed before injection to prevent retinal vascular occlusion secondary to raised intra-ocular pressure. Outcomes assessed were visual acuity, change in macular morphology, and complications.ResultsPatients presented promptly with delay between symptom onset and clinic review being 1.9±0.6 days (mean±SD). Treatment was delivered quickly with interval from presentation to treatment being 1.1±1.2 days. Symptom onset to treatment was 3.0±1.0 days. Subfoveal haemorrhage was effectively displaced in all patients. LogMAR visual acuity improved from 1.67±0.47 at presentation to 0.63±0.33 at final follow-up (P<0.0001), a mean of 7.9±4.8 months after treatment. Central retinal thickness improved from 658.1±174.2 μm at presentation to 316.6±142.4 μm at final follow-up (P=0.0028).ConclusionsEarly treatment of submacular haemorrhage using intravitreal tPA, C3F8, and anti-VEGF was effective in significantly improving visual acuity in this series of patients who presented soon after symptom onset. Treatment was well tolerated in this group of elderly and potentially frail patients.


A new endosymbiotic bivalve (Heterodonta: Galeommatoidea), from Pacific holothurians.
A fourth known species of Entovalva (Mollusca: Galeommatoidea), found in the esophagus of Holothuria spinifera and Holothuria leucospilota from Nha Trang Bay, Viet Nam, is described. Morphologic comparisons with the 3 previously described species are presented and the first DNA sequences for Entovalva are provided. Entovalva nhatrangensis, n. sp., differs from Entovalva mirabilis Voeltzkow, 1890 in its body shape, folded outer body epithelium, and lack of ovary in the foot. It differs from Entovalva (Cycladoconcha) amboinensis (Spärk, 1931) in its body shape and folded outer body epithelium. It differs from Entovalva lessonothuriae Kato, 1998, in shape of its foot. Two partial cytochrome oxidase I sequences for species diagnostic use have been submitted to GenBank.


Quantification of melamine absorption, distribution to tissues, and excretion by sheep.
Eight Döhne Merino rams were used to quantify apparent absorption, distribution to tissues, and excretion of dietary melamine in sheep. Two batches of concentrate pellets were made; one (CON) contained corn gluten meal with no detectable melamine and the other (MEL) contained corn gluten meal that was previously found to be highly contaminated with melamine at 15,117 mg/kg. The MEL pellets contained 1,149 mg/kg of melamine. During a 10-d adaptation period, all the animals received a forage-based diet supplemented with 600 g/d of the CON pellets. This was followed by an 8-d collection period during which 6 of the animals received MEL pellets and 2 received CON pellets. Melamine intake of sheep that received MEL pellets was 0.69 g/d. Blood samples were taken before first ingestion of MEL pellets on d 1 and again on d 3, 6, and 8 of the collection period for melamine and serum creatinine analyses. Feces and urine were collected quantitatively over the 8 d for proximate and melamine analyses. All the animals were slaughtered at the end of the trial, and samples of the LM, liver, kidneys, and abdominal fat were taken for melamine analysis. Data of the 2 sheep that received CON pellets for the duration of the trial confirmed that no melamine was detected in any of the samples, and no statistical analyses were performed on these data. The apparent digestibility or efficiency of absorption of ingested melamine was 76.7%. Melamine was detected in the urine, blood, muscle (LM), and fat tissue of all the sheep that received MEL pellets. Serum melamine concentrations reached 5.4 mg/kg on d 8 of the collection period, and the meat (LM) contained 9.6 mg/kg of melamine. Calculations on the partitioning of ingested melamine suggested that urine is the major excretion route accounting for 53.2%, whereas feces accounted for 23.3% of ingested melamine. Approximately 3.5% of the ingested melamine was detected in muscle. It was concluded that ingested melamine is highly absorbable from the small intestine and that a pathway exists for the distribution of dietary melamine to meat.


Comparison of modeled sampling strategies for estimation of dairy herd lameness prevalence and cow-level variables associated with lameness.
Monitoring herd lameness prevalence has utility for dairy producers and veterinarians in their efforts to reduce lameness, for animal welfare assessment programs, and for researchers. Locomotion scoring is a method used to quantify lameness and calculate prevalence. Because of the time necessary to locomotion score each cow in large dairy herds, a sampling strategy to determine herd lameness prevalence that allows scoring of fewer cows would be useful. Such a sampling strategy must be validated for accuracy compared with the lameness prevalence when all cows in a herd are locomotion scored. The purpose of this study was to assess 3 previously suggested methods of estimating lameness prevalence by strategic sampling of dairy herds. Sampling strategies tested included (1) sampling a calculated number of cows in the middle third of the milking parlor exit order for each pen, (2) sampling a calculated number of cows weighted across pens and distributed evenly within each pen, and (3) sampling all cows in the high production, low production, and hospital pens. Lactating cows on 5 dairy farms in Washington and Oregon (n=4,422) were locomotion scored using a 5-point scale to determine herd-level lameness prevalence (percentage with locomotion score ≥3). Milking parlor exit order, order in headlocks at the feed bunk within each pen, and breed were recorded for each cow. The number of days in lactation, milk production, and parity were collected from farm computer records. Pen grouping strategy for each farm was obtained by interview with farm management. Sampling strategies were modeled using the locomotion score data set for each herd. Estimates of lameness prevalence obtained from the milking parlor exit order sample and the sample distributed across pens were within 5 percentage points of the whole herd prevalence. The third strategy estimated the lameness prevalence within 5 percentage points on 4 farms, but overestimated prevalence on 1 farm. Pen-level prevalence obtained by locomotion score of all cows in the pen was variable and not reliably predictive of herd-level prevalence. Cows of Holstein breed, parity >1, and exiting the milking parlor in the last 20% of the pen had greater odds of lameness compared with other breeds, parities, and milking parlor exit order groups in a multivariate analysis. This study indicates that the sampling strategies using the middle of milking parlor exit order and a calculated sample distributed across the herd may be used to obtain an estimate of herd lameness prevalence.


Factors affecting chronic obstructive pulmonary disease early rehospitalization.
The Centers for Medicare and Medicaid Services has identified early rehospitalization of patients with chronic obstructive pulmonary disease (COPD) exacerbations as a performance measure for hospital care. We retrospectively reviewed patients with COPD who were admitted to University Medical Center, Lubbock, Texas, USA, between October 2010 and March 2011. There were 81 COPD patients with 103 hospitalizations. The mean age was 73.9 years. Pulmonary function tests using the Global initiative for chronic Obstructive Lung Disease criteria had been done in 36 patients (44.4%) and revealed 1 mild (2.8%), 7 moderate (19.4%), 20 severe (55.6%), and 8 very severe (22.2%) cases. Only 38.4% of the patients had prior influenza vaccine. Most patients were treated with antibiotics (81.8%) and corticosteroids (87.9%). The mean length of stay was 4.9 days, and 4 patients died. Most of the patients were discharged home (63.6%) with a median follow-up interval of 14 days. Thirty-two percent did not have long-acting bronchodilators and/or inhaled corticosteroids prescribed on discharge. There were 14 early rehospitalizations within 30 days. Logistic regression analysis indicated that a history of coronary artery disease (odds ratio (OR) 6.4, 95% confidence interval (CI) 1.1-37.4) and unilateral pulmonary infiltrates (OR 12.8, 95% CI 1.9-86.4) significantly increased the early rehospitalization rates. Acute exacerbations of COPD in patients with a history of ischemic heart disease or unilateral pulmonary infiltrates are at increased risk for early readmission. These risk factors should be identified during hospitalization; early follow-up or other interventions may reduce readmissions. Influenza vaccine, maintenance bronchodilators and/or inhaled corticosteroids, and pulmonary function tests were underused, and these standards of care should be provided to improve care.


Stimulus-dependent correlations and population codes.
The magnitude of correlations between stimulus-driven responses of pairs of neurons can itself be stimulus dependent. We examine how this dependence affects the information carried by neural populations about the stimuli that drive them. Stimulus-dependent changes in correlations can both carry information directly and modulate the information separately carried by the firing rates and variances. We use Fisher information to quantify these effects and show that, although stimulus-dependent correlations often carry little information directly, their modulatory effects on the overall information can be large. In particular, if the stimulus dependence is such that correlations increase with stimulus-induced firing rates, this can significantly enhance the information of the population when the structure of correlations is determined solely by the stimulus. However, in the presence of additional strong spatial decay of correlations, such stimulus dependence may have a negative impact. Opposite relationships hold when correlations decrease with firing rates.


Coexisting Primary Ovarian and Omental Hydatid Disease Mimicking an Ovarian Neoplasm: A Case Report.
Hydatid disease is a parasitic infection that most commonly affects the liver and lungs, although the disease can arise in any part of the body. Cysts may mimic many benign and malignant conditions. The diagnosis cannot be confirmed preoperatively in all cases. A 44-yr-old menopausal woman was admitted to the department of gynecology with complaints of abdominal distention. A fixed abdominopelvic mass was identified. Radiology revealed a 20-cm mass with branched septations and solid components. CA-125 level was 55 kU/L, and Risk of Malignancy Index-2 score was 880. These findings suggested the presence of an ovarian neoplasm, and laparotomy was performed. Cystic masses measuring 22 cm and 4 cm, originating from the omentum majus and left ovary, respectively, were found during surgery. Frozen-section analysis revealed hydatid disease. Infracolic omentectomy and total abdominal hysterectomy with bilateral salpingo-oopherectomy were performed. Results of a serum Echinococcus hemagglutination test performed immediately after surgery were negative. The patient was prescribed albendazole for 6 mo and discharged on the third postoperative day with no complaints. The incidence of hydatid disease in the female reproductive system is very rare; however, clinicians must be aware of this disease and take necessary precautions while operating because any spillage may lead to anaphylactic shock and increased risk of recurrence.


A rheumatology consultancy program with general practitioners in Catalonia, Spain.
To analyze the influence of a primary care rheumatology consultancy program on the number of referrals to the rheumatology unit (RU), the waiting times for new visits at the RU, and the satisfaction of the general practitioners (GPs) with respect to the RU. From September 2003 to August 2004 a consultancy program was carried out by 4 consultant rheumatologists of the RU and 117 GPs, consisting of biweekly clinical sessions in which cases were commented upon prior to referral and local clinical guidelines on 10 musculoskeletal disorders were discussed. Referral rates to the RU, 5-item GP satisfaction questionnaires (patient accessibility to the RU, information obtained after the visit, GP accessibility to the RU, communication between levels, and resolution capacity of the RU), waiting times, and number of patients waiting for a visit were analyzed. These variables were compared before and after the intervention. New referrals to the RU significantly diminished during the intervention year (1141 vs 1652 in the previous year, 5.5% patients referred vs 8.1% previous year; p < 0.0001). GP satisfaction improved significantly for all items (p < 0.0001). The waiting time for first non-urgent visit diminished from 7 months to 1 month (p < 0.01) during the intervention year, and the number of patients on the waiting list was reduced from 790 to 51 (p < 0.05). A consultancy program between rheumatologists of a RU and GPs of the same area showed improvement in GP satisfaction and reduced the number of referrals to the RU and the waiting times for new non-urgent visits.


Heat shock attenuates VEGF expression in three-dimensional myoblast sheets deteriorating therapeutic efficacy in heart failure.
Myoblast sheet transplantation is a promising novel treatment for ischemic heart failure. The aim of this study was to test the hypothesis that heat shock (HS) pre-treatment affects the angiogenic properties of myoblast sheets in vivo and in vitro. We studied HS preconditioning of L6 myoblast sheets in relation to their apoptosis, proliferation, and vascular endothelial growth factor (VEGF)-associated responses under normoxia and under hypoxia in vitro. In vivo evaluation of their therapeutic effect was performed with 60 male Wistar rats divided into 3 groups (20 each): sole left anterior descending (LAD) ligation (control); LAD ligation and non-conditioned sheet transplantation (L6 No-Shock); and LAD ligation and L6-heat shock conditioned sheet transplantation (L6 Heat-Shock). Left ventricular function was evaluated by echocardiography after 3, 10, and 28 days. Expression of HSP70/72 was strongly induced 24 hours after HS, and thereafter it decreased notably during 72 hours in hypoxia. Under normal growth conditions, HSP70/72 expression remained stable. HS delayed apoptosis-associated caspase-3 expression during 24-hour hypoxia compared to non-treated controls. However, VEGF expression reduced significantly in the heat shock pretreated sheets. Ejection fraction of the L6-myoblast HS pre-treatment group (L6 Heat-Shock) decreased gradually during follow-up, in the same pattern as the controls. However, these functional parameters improved in the L6-myoblast normal sheet group (L6 No-Shock) at the tenth day and remained significantly better. HS protects myoblast sheets from hypoxia-associated apoptosis in vitro, but reduces VEGF expression of the sheet, leading to lower therapeutic effect in heart failure.


Acute Stress Facilitates LTD Induction at Glutamatergic Synapses in the Hippocampal CA1 Region by Activating μ-Opioid Receptors on GABAergic Neurons.
Acute stress impairs recall memory through the facilitation of long-term depression (LTD) of hippocampal synaptic transmission. The endogenous opioid system (EOS) plays essential roles in stress-related emotional and physiological responses. Specifically, behavioral studies have shown that the impairment of memory retrieval induced by stressful events involves the activation of opioid receptors. However, it is unclear whether signaling mediated by μ-opioid receptors (μRs), one of the three major opioid receptors, participates in acute stress-related hippocampal LTD facilitation. Here, we examined the effects of a single elevated platform (EP) stress exposure on excitatory synaptic transmission and plasticity at the Schaffer collateral-commissural (SC) to CA1 synapses by recording electrically evoked field excitatory postsynaptic potentials and population spikes of hippocampal pyramidal neurons in anesthetized adult mice. EP stress exposure attenuated GABAergic feedforward and feedback inhibition of CA1 pyramidal neurons and facilitated low-frequency stimulation (LFS)-induced long-term depression (LTD) at SC-CA1 glutamatergic synapses. These effects were reproduced by exogenously activating μRs in unstressed mice. The specific deletion of μRs on GABAergic neurons (μRGABA) not only prevented the EP stress-induced memory impairment but also reversed the EP stress-induced attenuation of GABAergic inhibition and facilitation of LFS-LTD. Our results suggest that acute stress endogenously activates μRGABA to attenuate hippocampal GABAergic signaling, thereby facilitating LTD induction at excitatory synapses and eliciting memory impairments.


Termini restraining of small membrane proteins enables structure determination at near-atomic resolution.
Small membrane proteins are difficult targets for structural characterization. Here, we stabilize their folding by restraining their amino and carboxyl termini with associable protein entities, exemplified by the two halves of a superfolder GFP. The termini-restrained proteins are functional and show improved stability during overexpression and purification. The reassembled GFP provides a versatile scaffold for membrane protein crystallization, enables diffraction to atomic resolution, and facilitates crystal identification, phase determination, and density modification. This strategy gives rise to 14 new structures of five vertebrate proteins from distinct functional families, bringing a substantial expansion to the structural database of small membrane proteins. Moreover, a high-resolution structure of bacterial DsbB reveals that this thiol oxidoreductase is activated through a catalytic triad, similar to cysteine proteases. Overall, termini restraining proves exceptionally effective for stabilization and structure determination of small membrane proteins.


Snail1 induces epithelial-to-mesenchymal transition and tumor initiating stem cell characteristics.
Tumor initiating stem-like cells (TISCs) are a subset of neoplastic cells that possess distinct survival mechanisms and self-renewal characteristics crucial for tumor maintenance and propagation. The induction of epithelial-mesenchymal-transition (EMT) by TGFβ has been recently linked to the acquisition of TISC characteristics in breast cancer. In HCC, a TISC and EMT phenotype correlates with a worse prognosis. In this work, our aim is to elucidate the underlying mechanism by which cells acquire tumor initiating characteristics after EMT. Gene and protein expression assays and Nanog-promoter luciferase reporter were utilized in epithelial and mesenchymal phenotype liver cancer cell lines. EMT was analyzed with migration/invasion assays. TISC characteristics were analyzed with tumor-sphere self-renewal and chemotherapy resistance assays. In vivo tumor assay was performed to investigate the role of Snail1 in tumor initiation. TGFβ induced EMT in epithelial cells through the up-regulation of Snail1 in Smad-dependent signaling. Mesenchymal liver cancer post-EMT demonstrates TISC characteristics such as tumor-sphere formation but are not resistant to cytotoxic therapy. The inhibition of Snail1 in mesenchymal cells results in decreased Nanog promoter luciferase activity and loss of self-renewal characteristics in vitro. These changes confirm the direct role of Snail1 in some TISC traits. In vivo, the down-regulation of Snail1 reduced tumor growth but was not sufficient to eliminate tumor initiation. In summary, TGFβ induces EMT and TISC characteristics through Snail1 and Nanog up-regulation. In mesenchymal cells post-EMT, Snail1 directly regulates Nanog expression, and loss of Snail1 regulates tumor growth without affecting tumor initiation.


Global DNA methylation changes and differential gene expression in Anaplasma phagocytophilum-infected human neutrophils.
Anaplasma phagocytophilum is an obligate intracellular prokaryotic pathogen that both infects and replicates within human neutrophils. The bacterium represses multiple antimicrobial functions while simultaneously increasing proinflammatory functions by reprogramming the neutrophil genome. Previous reports show that many observed phenotypic changes are in part explained by altered gene transcription. We recently identified that large chromosomal regions of the neutrophil genome are differentially expressed during A. phagocytophilum infection. Because of this, we sought to determine whether gene expression programs altered by infection were the result of changes in the host neutrophil DNA methylome. Within 24 h of infection, marked increases in DNA methylation were observed genome-wide as compared with mock-infected controls and pharmacologic inhibition of DNA methyltransferases resulted in decreased bacterial growth. New regions of DNA methylation were enriched at intron and exon junctions; however, intragenic methylation did not correlate with altered gene expression. In contrast, intergenic DNA methylation was associated with A. phagocytophilum-induced gene expression changes. Within the major histocompatibility complex locus on chromosome 6, a region with marked changes in infection-induced differential gene expression, new regions of methylation were localized to boundaries of active and inactive chromatin. These data strongly suggest that A. phagocytophilum infection, in addition to altering histone structure, alters DNA methylation and the epigenome of its host cell to promote survival and replication, providing evidence that such bacterial infection can radically alter the epigenome of its host cell.


Using Hybrid Change Strategies to Improve the Patient Experience in Outpatient Specialty Care.
The emerging changes in healthcare impose significant burdens on integrated outpatient specialty services with respect to setting patient expectations, handling outside medical records; and coordinating specialty appointments scheduling. Moreover, because of the evolution of the electronic health record and its widespread use, it is critical that patient and physician interaction is maintained and clerical tasks are minimized. In the context of increased government regulation, declining reimbursement, and the rise of new payment models, outpatient practices need to be reimagined so that they are more efficient for the patient and the provider. The redesign of integrated outpatient specialty services can be accomplished only through teamwork, innovation, and efficient use of technology. To address these challenges, the Department of Medicine at Mayo Clinic in Scottsdale, Arizona, implemented an ideal practice design initiative that leveraged a hybrid set of change strategies. The change strategy, which was initiated after examination of current practices and design options, engaged key stakeholders and patients. A number of enablers and barriers to adoption were identified as a result of the implementation experience.


α2-Adrenergic Receptor and Isoflurane Modulation of Presynaptic Ca2+ Influx and Exocytosis in Hippocampal Neurons.
Evidence indicates that the anesthetic-sparing effects of α2-adrenergic receptor (AR) agonists involve α2A-AR heteroreceptors on nonadrenergic neurons. Since volatile anesthetics inhibit neurotransmitter release by reducing synaptic vesicle (SV) exocytosis, the authors hypothesized that α2-AR agonists inhibit nonadrenergic SV exocytosis and thereby potentiate presynaptic inhibition of exocytosis by isoflurane. Quantitative imaging of fluorescent biosensors of action potential-evoked SV exocytosis (synaptophysin-pHluorin) and Ca influx (GCaMP6) were used to characterize presynaptic actions of the clinically used α2-AR agonists dexmedetomidine and clonidine, and their interaction with isoflurane, in cultured rat hippocampal neurons. Dexmedetomidine (0.1 μM, n = 10) or clonidine (0.5 μM, n = 8) inhibited action potential-evoked exocytosis (54 ± 5% and 59 ± 8% of control, respectively; P < 0.001). Effects on exocytosis were blocked by the subtype-nonselective α2-AR antagonist atipamezole or the α2A-AR-selective antagonist BRL 44408 but not by the α2C-AR-selective antagonist JP 1302. Dexmedetomidine inhibited exocytosis and presynaptic Ca influx without affecting Ca coupling to exocytosis, consistent with an effect upstream of Ca-exocytosis coupling. Exocytosis coupled to both N-type and P/Q-type Ca channels was inhibited by dexmedetomidine or clonidine. Dexmedetomidine potentiated inhibition of exocytosis by 0.7 mM isoflurane (to 42 ± 5%, compared to 63 ± 8% for isoflurane alone; P < 0.05). Hippocampal SV exocytosis is inhibited by α2A-AR activation in proportion to reduced Ca entry. These effects are additive with those of isoflurane, consistent with a role for α2A-AR presynaptic heteroreceptor inhibition of nonadrenergic synaptic transmission in the anesthetic-sparing effects of α2A-AR agonists.


Looking into the "Garden of the Hesperides": new drugs for hepatocellular carcinoma.
Advanced hepatocellular carcinoma remains an entity that is hard to approach therapeutically and has shown disappointing results in terms of survival. For many years, the only accepted option for this setting was the use of a multikinase inhibitor, sorafenib. Nevertheless, through the years, a deeper knowledge has arisen about how pathogenic pathways correlate with hepatocellular carcinoma. In this review, we provide an update of the most recent data regarding new agents under investigation and new possible targets for future treatments.


Travis and Arms revisited: a second look at a widely used bioconcentration algorithm.
In 1988, Travis and Arms reviewed the literature and collected data to develop a relationship between the octanol-water partition coefficient (Kow) and the uptake of organic compounds into milk and beef (Travis and Arms, 1988). These equations have been utilized for predicting biotransfer factors for organic chemicals when empirical data are lacking. During the external peer review of the draft US Environmental Protection Agency (EPA) guidance entitled Human Health Risk Assessment Protocol for Hazardous Waste Combustion Facilities (US EPA, 1998) and the development of Superfund's Ecological Soil Screening Levels (US EPA, 2000b), questions challenging the derivation and use of these equations were raised. The primary questions raised were: 1) Are the equations presented in Travis and Arms (1988) for the estimation of transfer of organic compounds from contaminated feed to beef and milk technically valid and reproducible? If so, (2) are the equations appropriate across the entire log Kow range? For these reasons, this study was undertaken to validate the original Travis and Arms equations, to review more recent literature, and. if appropriate, to add to the original Travis and Arms data set to obtain updated equations. This paper presents an evaluation of the original Travis and Arms equations, limitations to their use, and steps to reduce uncertainties associated with their use by updating with more current literature.


Embolic Doppler ultrasound signal detection via fractional Fourier transform.
Computerized analysis of Doppler ultrasound signals can aid early detection of asymptomatic circulating emboli. For analysis, physicians use informative features extracted from Doppler ultrasound signals. Time -frequency analysis methods are useful tools to exploit the transient like signals such as Embolic signals. Detection of discriminative features would be the first step toward automated analysis of embolic Doppler ultrasound signals. The most problematic part of setting up emboli detection system is to differentiate embolic signals from confusing similar wave-like patterns such as Doppler speckle and artifacts caused by tissue movement, probe tapping, speaking etc. In this study, discrete version of fractional Fourier transform is presented as a solution in the detection of emboli in digitized Doppler ultrasound signals. An accurate set of parameters are extracted using short time Fourier transform and fractional Fourier transform and the results are compared to reveal detection quality. Experimental results prove the efficiency of fractional Fourier transform in which discriminative features becomes more evident.


Assessing Latin America's Progress Toward Achieving Universal Health Coverage.
Two commonly used metrics for assessing progress toward universal health coverage involve assessing citizens' rights to health care and counting the number of people who are in a financial protection scheme that safeguards them from high health care payments. On these metrics most countries in Latin America have already "reached" universal health coverage. Neither metric indicates, however, whether a country has achieved universal health coverage in the now commonly accepted sense of the term: that everyone--irrespective of their ability to pay--gets the health services they need without suffering undue financial hardship. We operationalized a framework proposed by the World Bank and the World Health Organization to monitor progress under this definition and then constructed an overall index of universal health coverage achievement. We applied the approach using data from 112 household surveys from 1990 to 2013 for all twenty Latin American countries. No country has achieved a perfect universal health coverage score, but some countries (including those with more integrated health systems) fare better than others. All countries except one improved in overall universal health coverage over the time period analyzed.


More images that yet fresh images Beget.
In this issue of Immunity, Dong et al. (2009) describe the protein crystal structure heterodimer of tapasin and ERp57, which helps visualize the function of these proteins in loading of peptide antigens onto MHC class I molecules as part of the peptide loading complex within the endoplasmic reticulum.


Reproductive Potential of Field-collected Populations of Cimex lectularius L. and the Cost of Traumatic Insemination.
Egg production was compared among three field-collected bed bug strains over the course of 13 feeding/oviposition cycles, each of which lasted ~10 days. No significant differences were found among bed bug strains in the mean number of eggs/female/day (~1.0 egg). However, significant differences were found among strains in their patterns of egg production throughout the study period. Specifically, differences were observed in the timing of peak egg production and the rapidity of egg production decline among the three strains. Egg production was also quantified for female bed bugs that were subjected to single or multiple traumatic insemination events over a period of six feeding/oviposition cycles. Significant differences were found in egg production between females exposed to single and multiple inseminations. Females mated only once produced 83.8 ± 4.5 (mean ± SE) eggs over six feeding cycles. Females exposed to multiple inseminations produced 61.0 ± 3.1 eggs, indicating that multiple traumatic inseminations may reduce female fecundity by as much as 27%. This study is the first to suggest that, in a new infestation (first ~6 weeks), a solitary, singly-mated female with access to regular blood meals is capable of producing greater numbers of offspring than the same female in the presence of a male.


Evaluation of Eggplant Rootstocks and Pepper Varieties for Potential Resistance to Isolates of Phytophthora capsici from Michigan and New York.
Phytophthora capsici is a soilborne pathogen of major economic importance in pepper, and of less importance in tomato and eggplant production. As soil fumigation becomes more expensive and limited, and fungicide insensitivity of P. capsici isolates becomes more prevalent, grafting is quickly becoming an industry-favored method to control soilborne diseases. Greenhouse experiments were performed to evaluate an eggplant cultivar (Classic), two eggplant lines (EG195, EG203), a pepper line (CM334), and three pepper cultivars (Paladin, Camelot, and Red Knight) for root rot resistance to 14 P. capsici isolates. The isolates showed various degrees of virulence between pepper and eggplant in both experiments. Both eggplant and one pepper lines showed moderate resistance to the most virulent isolates tested in experiment one. The partially resistant pepper cultivar, Paladin, was significantly more susceptible than CM334 and the eggplant lines, but was still resistant to most isolates. In the second experiment, the eggplant cultivar Classic and the susceptible pepper cultivar Red Knight were both susceptible to most isolates tested, while EG203 and EG195 were resistant to most isolates. The two eggplant breeding lines, EG195 and EG203, showed moderate resistance to all isolates tested in both experiments. This is the first reported evaluation of eggplant resistance to P. capsici. Further research is warranted to test eggplant lines EG195 and EG203 for resistance to a wide range of soilborne pests and to evaluate their usefulness as P. capsici-resistant rootstocks for peppers, tomatoes, and eggplants.


Research, policy, and the National Health Service.
The National Health Service is a system designed to bring about a rational use and distribution of resources yet which largely ignores the contribution of the research community. With a relatively closed health policy arena, there are few customers for policy-oriented research. With responsibility for funding research concentrated at the center and responsibility for delivering services at the periphery, the research community finds itself in limbo. In comparison to both the U.S. and Canada, Britain therefore offers an example of research both underfinanced and undervalued. However, research has made some significant contributions in areas where there has been a perceived use for its findings to support service developments. And the changes now being introduced in Britain's NHS are likely to create a new market for research as the system adopts some North American ideas and becomes less consensual and more pluralistic.


Nek6 and Hif-1α cooperate with the cytoskeletal gateway of drug resistance to drive outcome in serous ovarian cancer.
Hypoxia selects the most aggressive and drug-resistant clones in solid malignancies. One of the pivotal transcription factors induced by hypoxia is Hif-1α. However, in serous ovarian cancer (SEOC), Hif-1α expression is not a prognostic biomarker. This study aims to assess the hypothesis that the serine-threonine kinase Nek6 functions as a downstream effector cooperating with Hif-1α in driving ovarian cancer aggressiveness. Nek6 was overexpressed and Hif-1α was silenced in A2780 cells. Nek6 was also stably silenced in Hey cells. The dependence of Nek6 expression on Hif-1α was assayed as a function of hypoxic growth conditions. Nek6 interaction with the cytoskeletal gateway of drug resistance was investigated with far western blot. The co-expression of NEK6, HIF1A, TUBB3 and GBP1 transcripts was quantified with qPCR in two cohorts of SEOC patients (346 locally treated patients and 344 from the TCGA dataset). Nek6 expression is induced by hypoxia in a Hif-1α dependent fashion. Nek6 directly interacts with GBP-1, thus being a component of the cytoskeletal gateway of drug resistance. Nek6 overexpression increases and silencing decreases the anchorage-independent growth of cultured cells. In SEOC patients, NEK6 expression is significantly correlated with HIF1A. Co-expression of NEK6, HIF1A, TUBB3 and GBP1 transcripts identifies a subset of SEOC patients characterized by poor outcome and drug resistance. This study demonstrates the functional relevance of Nek6 in the context of the adaptive response to hypoxia in SEOC. This finding may help identify a sub-population of patients at high risk of relapse to standard first-line chemotherapy.


Comparative physical mapping between Oryza sativa (AA genome type) and O. punctata (BB genome type).
A comparative physical map of the AA genome (Oryza sativa) and the BB genome (O. punctata) was constructed by aligning a physical map of O. punctata, deduced from 63,942 BAC end sequences (BESs) and 34,224 fingerprints, onto the O. sativa genome sequence. The level of conservation of each chromosome between the two species was determined by calculating a ratio of BES alignments. The alignment result suggests more divergence of intergenic and repeat regions in comparison to gene-rich regions. Further, this characteristic enabled localization of heterochromatic and euchromatic regions for each chromosome of both species. The alignment identified 16 locations containing expansions, contractions, inversions, and transpositions. By aligning 40% of the punctata BES on the map, 87% of the punctata FPC map covered 98% of the O. sativa genome sequence. The genome size of O. punctata was estimated to be 8% larger than that of O. sativa with individual chromosome differences of 1.5-16.5%. The sum of expansions and contractions observed in regions >500 kb were similar, suggesting that most of the contractions/expansions contributing to the genome size difference between the two species are small, thus preserving the macro-collinearity between these species, which diverged approximately 2 million years ago.


Morphology dependent adsorption of methylene blue on trititanate nanoplates and nanotubes prepared by the hydrothermal treatment of TiO2.
The adsorption properties of two nanomorphologies of trititanate, nanotubes (TiNT) and plates (TiNP), prepared by the hydrothermal reaction of concentrated NaOH with different phases of TiO2, were examined. It was found that the capacity for both morphologies towards methylene blue (MB), an ideal pollutant, was extremely high, with the TiNP having a capacity of 130 mg/g, higher than the TiNT, whose capacity was 120 mg/g at 10 mg/L MB concentration. At capacity, the well-dispersed powders deposit on the floor of the reaction vessel. The two morphologies had very different structural and adsorption properties. TiNT with high surface area and pore volume exhibited exothermic monolayer adsorption of MB. TiNP with low surface area and pore volume yielded a higher adsorption capacity through endothermic multilayer adsorption governed by pore diffusion. TiNP exhibited a higher negative surface charge of -23 mV, compared to -12 mV for TiNT. The adsorption process appears to be an electrostatic interaction, with the cationic dye attracted more strongly to the nanoplates, resulting in a higher adsorption capacity and different adsorption modes. We believe this simple, low cost production of high capacity nanostructured adsorbent material has potential uses in wastewater treatment.


Ultrasensitive detection of mRNA extracted from cancerous cells achieved by DNA rotaxane-based cross-rolling circle amplification.
An ultrasensitive and highly selective method for polymerase chain reaction-free (PCR-free) messenger RNA (mRNA) expression profiling is developed through a novel cross-rolling circle amplification (C-RCA) process based on DNA-rotaxane nanostructures. Two species of DNA pseudorotaxane (DPR) superstructures (DPR-I and DPR-II) are assembled by threading a linear DNA rod through a double-stranded DNA (dsDNA) ring containing two single-stranded gaps. In this assay, cDNA that is specific for β-actin (ACTB) mRNA is taken as a model analyte. Upon the introduction of the target cDNA, the cDNA and the biotin-modified primer are hybridized to the single-stranded regions of the DNA rod and the gap-ring, respectively. As a result, the DPR-I dethreads into free DNA macrocycle and a dumbbell-shaped DNA nanostructure. In the presence of DNA polymerase/dNTPs, two release-DNA on the DPR-I are replaced by polymerase with strand-displacement activity, which can act as the input of the DPR-II to trigger the dethreading of DPR-II and the RCA reaction, releasing another two specified release-DNA strands those in turn serve as the "mimic cDNA" for DPR-I. The C-RCA reaction then proceeds autonomously. To overcome the high background induced by hemin itself, the biotinylated rolling circle products are captured by streptavidin-coated MNPs, achieving a detection limit as low as 0.1 zmol cDNA. The assay also exhibits an excellent selectivity due to its unique DNA nanostructure fabricated through base pairing hybridization. The ACTB mRNA expression in mammary cancer cells (MCF-7) is successfully detected.


Pharmacokinetics of clobazam oral soluble film.
Clobazam oral soluble film (COSF) is a novel dosage form under development for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome. The present study was undertaken to assess the pharmacokinetics of clobazam administered as single doses of COSF 20 and 10 mg compared with clobazam tablets (CTAB) 20 and 10 mg in healthy adults. A secondary objective was to assess the safety and tolerability of single doses of COSF 20 and 10 mg. A total of 51 adult volunteers were enrolled in a single-dose, open-label, randomized four-sequence, four-period, crossover study with treatments (A) COSF 20 mg, (B) CTAB 20 mg, (C) COSF 10 mg, and (D) CTAB 10 mg. Pharmacokinetic sampling for clobazam and N-desmethylclobazam was carried out until 21 days postdose with a 28-day washout. Subjects were monitored for adverse events (AEs) throughout the study. Visual inspections of the administration site were performed before and after COSF administration to monitor for mucosal irritation. COSF at single doses of 10 and 20 mg was bioequivalent to CTAB at equivalent doses for both clobazam and its active metabolite N-desmethylclobazam. The pharmacokinetics of both formulations was dose-proportional at doses of 10 and 20 mg. The number of AEs and the number of subjects experiencing AEs were dose-related across the treatment groups, with somnolence the most common event. None of these events was severe or serious, and most were mild. There was no evidence for local irritation at the administration site following COSF. COSF is a novel clobazam dosage form that is bioequivalent to CTAB. Because of its ease of administration, COSF may be expected to improve adherence, reduce likelihood of dosing error, and provide more accurate dosing than formulations of clobazam that are currently available.


Integration of root phenes for soil resource acquisition.
Suboptimal availability of water and nutrients is a primary limitation to plant growth in terrestrial ecosystems. The acquisition of soil resources by plant roots is therefore an important component of plant fitness and agricultural productivity. Plant root systems comprise a set of phenes, or traits, that interact. Phenes are the units of the plant phenotype, and phene states represent the variation in form and function a particular phene may take. Root phenes can be classified as affecting resource acquisition or utilization, influencing acquisition through exploration or exploitation, and in being metabolically influential or neutral. These classifications determine how one phene will interact with another phene, whether through foraging mechanisms or metabolic economics. Phenes that influence one another through foraging mechanisms are likely to operate within a phene module, a group of interacting phenes, that may be co-selected. Examples of root phene interactions discussed are: (1) root hair length × root hair density, (2) lateral branching × root cortical aerenchyma (RCA), (3) adventitious root number × adventitious root respiration and basal root growth angle (BRGA), (4) nodal root number × RCA, and (5) BRGA × root hair length and density. Progress in the study of phenes and phene interactions will be facilitated by employing simulation modeling and near-isophenic lines that allow the study of specific phenes and phene combinations within a common phenotypic background. Developing a robust understanding of the phenome at the organismal level will require new lines of inquiry into how phenotypic integration influences plant function in diverse environments. A better understanding of how root phenes interact to affect soil resource acquisition will be an important tool in the breeding of crops with superior stress tolerance and reduced dependence on intensive use of inputs.


Mode of Action of Clofazimine and Combination Therapy with Benzothiazinones against Mycobacterium tuberculosis.
Clofazimine (CZM) is an antileprosy drug that was recently repurposed for treatment of multidrug-resistant tuberculosis. In Mycobacterium tuberculosis, CZM appears to act as a prodrug, which is reduced by NADH dehydrogenase (NDH-2), to release reactive oxygen species upon reoxidation by O2. CZM presumably competes with menaquinone (MK-4), a key cofactor in the mycobacterial electron transfer chain, for its reduction by NDH-2. We studied the effect of MK-4 supplementation on the activity of CZM against M. tuberculosis and found direct competition between CZM and MK-4 for the cidal effect of CZM, against nonreplicating and actively growing bacteria, as MK-4 supplementation blocked the drug's activity against nonreplicating bacteria. We demonstrated that CZM, like bedaquiline, is synergistic in vitro with benzothiazinones such as 2-piperazino-benzothiazinone 169 (PBTZ169), and this synergy also occurs against nonreplicating bacteria. The synergy between CZM and PBTZ169 was lost in an MK-4-rich medium, indicating that MK-4 is the probable link between their activities. The efficacy of the dual combination of CZM and PBTZ169 was tested in vivo, where a great reduction in bacterial load was obtained in a murine model of chronic tuberculosis. Taken together, these data confirm the potential of CZM in association with PBTZ169 as the basis for a new regimen against drug-resistant strains of M. tuberculosis.


Personality Structure Among Centenarians: The Georgia Centenarian Study.
BACKGROUND/STUDY CONTEXT: We demonstrate that observer-rated factor structure of personality in centenarians is congruent with the normative structure. Prevalence of cognitive impairment, which has previously been linked to changes in personality in younger samples, is high in this age group, requiring observer ratings to obtain valid data in a population-based context. Likewise, the broad range of cognitive functioning necessitates synthesis of results across multiple measures of cognitive performance. Data from 161 participants in the Georgia Centenarian Study (GCS; MAge = 100.3 years, 84% women, 20% African American, 40% community-dwelling, 30% low cognitive functioning) support strong overall correspondence with reference structure (full sample: .94; higher cognitive functioning: .94; lower cognitive functioning: .90). Centenarians with lower cognitive functioning are higher on neuroticism and lower on openness to experience, agreeableness, and conscientiousness. Facet-level differences (higher N1-N6: anxiety, hostility, depression, self-consciousness, impulsiveness, vulnerability to stress; lower E1: warmth; lower O4-O6: actions, ideas, values; lower A1, A3, A4: trust, altruism, compliance; C1, C5: competence, self-discipline) are also observed. Multivariate factor-level models indicate only neuroticism of the five broad factors predicts membership in cognitively impaired group; facet-level models showed that lower-order scales from three of the five domains were significant. Centenarians with higher self-consciousness (N4), impulsiveness (N5), and deliberation (C6) but lower ideas (O5), compliance (A4), and self-discipline (C5) were more likely to be in the lower cognitive functioning category. Results present first normative population-based data for personality structure in centenarians and offer intriguing possibilities for the role of personality in cognitive impairment centered on neuroticism.


Localization of the active site of HIV-1 reverse transcriptase-associated RNase H domain on a DNA template using site-specific generated hydroxyl radicals.
Reverse transcriptase (RT)-associated ribonuclease H (RNase H) can cleave both the RNA template of DNA/RNA hybrids as well as double-stranded (ds) RNA. This report shows that human immunodeficiency virus (HIV)-RT can also cleave the template strand of dsDNA when Mg2+ is replaced by Fe2+ in the RNase H active site of HIV-RT. The cleavage mechanisms as well as the positions of the cut vary depending on whether RNA or DNA is used. While DNA is cleaved 17 base positions upstream of the primer 3'-end, RNA is cleaved 18 base positions upstream. Competition experiments show that Fe2+ replaces the catalytically active Mg2+ of RT-associated RNase H. The bound Fe2+ is the source of locally generated OH-radicals that cleave the most proximate base in the DNA. Electrophoretic mobility studies of the cleaved fragments suggest that DNA is cleaved by an oxidative mechanism, while RNA is cleaved by an enzymatic mechanism which is indistinguishable from the Mg2+-dependent cleavage. The Fe2+-dependent cuts can be used to trace the active site of RT-associated RNase H on dsDNA as well as on dsRNA and DNA/RNA hybrids. The observed 1 base difference in the cleavage positions on DNA and RNA templates can be attributed to conformational differences of the bound nucleic acids. We suggest that the lower pitch of dsRNA and DNA/RNA hybrids compared with dsDNA permits accommodation of an additional base pair in the region between the primer 3'-end and the Fe2+-dependent cleavage position at the RNase H active site.


Anti-thyroid methimazole in an acidosis-induced retinopathy rat model of retinopathy of prematurity.
Methimazole (MMI), an anti-thyroid drug known to reduce serum levels of L-thyroxine (T4) and insulin-like growth factor-1 (IGF-1), has been previously reported to increase the incidence of neovascularization (NV) in an oxygen-induced retinopathy (OIR) model of retinopathy of prematurity (ROP) in rats. We investigated the effect of MMI on the incidence and severity of NV in a non-oxygen-induced model of ROP, acidosis-induced retinopathy (AIR). Newborn Sprague Dawley rats were raised in expanded litters of 25 in room air for four or ten days under one of the two following conditions: (1) Our established model of AIR (acidosis via NH4Cl gavage (10 mmol/kg) twice daily from days 2 to 7, followed by two days of recovery) or (2) MMI (given as a 0.1% solution to nursing mothers) in the above AIR model. Left eyes were fixed, and retinas were dissected and ADPase-stained. Flat mounted retinas were graded in a masked manner for presence and severity of NV, and retinal vascular areas were quantified. Serum IGF-1 and T4 concentrations were measured by radioimmunoassay on days 4 and 10. Arterial blood pH measurements were performed on day 4. The incidence and severity of NV were similar between AIR and MMI-AIR rats (incidence: 24% and 33%). Serum IGF-1 concentrations in 10 day MMI-AIR rats were significantly lower than untreated non-acidotic controls (medians: 158 ng/ml and 207 ng/ml; p=0.03). Serum IGF-1 concentrations were similar between 10 day AIR rats and untreated non-acidotic controls (medians: 189 ng/ml and 207 ng/ml; p>0.9). MMI does not increase the incidence or severity of NV in an AIR neonatal rat model of ROP. Although serum IGF-1 has been considered permissive for NV in immature retinas, supranormal concentrations of serum IGF-1 may not be necessary for abnormal retinal angiogenesis. Further studies are warranted on the roles of serum IGF-1 and L-thyroxine in the pathogenesis of ROP.


Clinical associations between thyroid and liver diseases.
The liver has an important role in thyroid hormone metabolism and the level of thyroid hormones is also important to normal hepatic function and bilirubin metabolism. Besides the associations between thyroid and liver diseases of an autoimmune nature, such as that between primary biliary cirrhosis and hypothyroidism, thyroid diseases are frequently associated with liver injuries or biochemical test abnormalities. For example, thyroid diseases may be associated with elevation of alanine aminotransferase and alkaline phosphatase, which is mainly of bone origin, in hyperthyroidism and aspartate aminotransferase in hypothyroidism. Liver diseases are also frequently associated with thyroid test abnormalities or dysfunctions, particularly elevation of thyroxine-binding globulin and thyroxine. Hepatitis C virus infection has been connected with thyroid abnormalities. In addition, antithyroid drug therapy may result in hepatitis, cholestasis or transient subclinical hepatotoxicity, whereas interferon (IFN) therapy in liver diseases may also induce thyroid dysfunctions. These thyroid-liver associations may cause diagnostic confusions. Neglect of these facts may result in over of under diagnosis of associated liver or thyroid diseases and thereby cause errors in patient care. It is suggested to measure free thyroxine (FT4) and thyroid-stimulating hormone (TSH) which are usually normal in euthyroid patients with liver disease, to rule out or rule in coexistent thyroid dysfunctions, and consider the possibility of thyroid dysfunctions in any patients with unexplained liver biochemical test abnormalities. It is also advisable to monitor patients with autoimmune liver disease or those receiving IFN therapy for the development of thyroid dysfunctions, and patients receiving antithyroid therapy for the development of hepatic injuries.


Csu pili dependent biofilm formation and virulence of Acinetobacter baumannii.
Acinetobacter baumannii has emerged as one of the most common extensive drug-resistant nosocomial bacterial pathogens. Not only can the bacteria survive in hospital settings for long periods, but they are also able to resist adverse conditions. However, underlying regulatory mechanisms that allow A. baumannii to cope with these conditions and mediate its virulence are poorly understood. Here, we show that bi-stable expression of the Csu pili, along with the production of poly-N-acetyl glucosamine, regulates the formation of Mountain-like biofilm-patches on glass surfaces to protect bacteria from the bactericidal effect of colistin. Csu pilus assembly is found to be an essential component of mature biofilms formed on glass surfaces and of pellicles. By using several microscopic techniques, we show that clinical isolates of A. baumannii carrying abundant Csu pili mediate adherence to epithelial cells. In addition, Csu pili suppressed surface-associated motility but enhanced colonization of bacteria into the lungs, spleen, and liver in a mouse model of systemic infection. The screening of c-di-GMP metabolizing protein mutants of A. baumannii 17978 for the capability to adhere to epithelial cells led us to identify GGDEF/EAL protein AIS_2337, here denoted PdeB, as a major regulator of Csu pili-mediated virulence and biofilm formation. Moreover, PdeB was found to be involved in the type IV pili-regulated robustness of surface-associated motility. Our findings suggest that the Csu pilus is not only a functional component of mature A. baumannii biofilms but also a major virulence factor promoting the initiation of disease progression by mediating bacterial adherence to epithelial cells.


Band gap modulation of SrTiO3 upon CO2 adsorption.
Herein, CO2 chemisorption on SrTiO3(001) surfaces is studied using ab initio calculations to establish new chemical sensing mechanisms. It was found that CO2 adsorption opens the band gap of the material. However, the mechanisms are different: the CO2 adsorption on the TiO2-terminated surface neutralizes the surface states at the valence band (VB) maximum, whereas for the SrO-terminated surface it suppresses the conduction band (CB) minimum. For the TiO2-terminated surface, the effect is explained by the passivation of dangling bonds, whereas for the SrO-terminated surface, the suppression is caused by surface relaxation. Modulation of the VB states implies a more direct change in charge distribution, and thus, the induced change in the band gap is more prominent at the TiO2 termination. Further, it has been shown that both CO2 adsorption energy and surface band gap are strongly dependent on CO2 coverage, suggesting that the observed effect can be utilized in sensing applications for a wide range of CO2 concentrations.


OsATG7 is required for autophagy-dependent lipid metabolism in rice postmeiotic anther development.
In flowering plants, the tapetum, the innermost layer of the anther, provides both nutrient and lipid components to developing microspores, pollen grains, and the pollen coat. Though the programmed cell death of the tapetum is one of the most critical and sensitive steps for fertility and is affected by various environmental stresses, its regulatory mechanisms remain mostly unknown. Here we show that autophagy is required for the metabolic regulation and nutrient supply in anthers and that autophagic degradation within tapetum cells is essential for postmeiotic anther development in rice. Autophagosome-like structures and several vacuole-enclosed lipid bodies were observed in postmeiotic tapetum cells specifically at the uninucleate stage during pollen development, which were completely abolished in a retrotransposon-insertional OsATG7 (autophagy-related 7)-knockout mutant defective in autophagy, suggesting that autophagy is induced in tapetum cells. Surprisingly, the mutant showed complete sporophytic male sterility, failed to accumulate lipidic and starch components in pollen grains at the flowering stage, showed reduced pollen germination activity, and had limited anther dehiscence. Lipidomic analyses suggested impairment of editing of phosphatidylcholines and lipid desaturation in the mutant during pollen maturation. These results indicate a critical involvement of autophagy in a reproductive developmental process of rice, and shed light on the novel autophagy-mediated regulation of lipid metabolism in eukaryotic cells.


An Alternative Framework for Defining Mediation.
The present article provides an alternative framework for evaluating mediated relationships. From this perspective. a mediated process is a chain reaction, beginning with an independent variable that affects a mediator that in turn affects an outcome. The definition of mediation offered here, presented for stage sequences, states three conditions for establishing mediation: (a) the independent variable affects the probability of the sequence no mediator to mediator to outcome; (b) the independent variable affects the probability of a transition into the mediator stage; (c) the mediator affects the probability of a transition into the outcome stage at every level of the independent variable. This definition of mediation is compared and contrasted with the well-known definition of mediation for continuous variables discussed in Baron and Kenny (1986), Judd and Kenny (1981), and Kenny, Kashy, and Bolger (1997). The definition presented in this article emphasizes the intraindividual, time-ordered nature of mediation.


Development of a covalent inhibitor of gut bacterial bile salt hydrolases.
Bile salt hydrolase (BSH) enzymes are widely expressed by human gut bacteria and catalyze the gateway reaction leading to secondary bile acid formation. Bile acids regulate key metabolic and immune processes by binding to host receptors. There is an unmet need for a potent tool to inhibit BSHs across all gut bacteria to study the effects of bile acids on host physiology. Here, we report the development of a covalent pan-inhibitor of gut bacterial BSHs. From a rationally designed candidate library, we identified a lead compound bearing an alpha-fluoromethyl ketone warhead that modifies BSH at the catalytic cysteine residue. This inhibitor abolished BSH activity in conventional mouse feces. Mice gavaged with a single dose of this compound displayed decreased BSH activity and decreased deconjugated bile acid levels in feces. Our studies demonstrate the potential of a covalent BSH inhibitor to modulate bile acid composition in vivo.


Cross-talk between calcium and cAMP-dependent intracellular signaling pathways. Implications for synergistic secretion in T84 colonic epithelial cells and rat pancreatic acinar cells.
Treatment of various cells with combinations of agents that increase either cAMP or cytosolic calcium can lead to synergistic responses. This study examined interactions, or cross-talk, between these two intracellular messengers and its implication for signaling in two secretory cell types, T84 human colonic epithelial cells and rat pancreatic acinar cells. T84 cell chloride secretion was measured in Ussing chambers. Acinar cell activation was monitored as amylase secretion. Cytosolic calcium was assessed via fura-2 microfluorimetry. A cell-permeant analogue of cAMP synergistically enhanced secretory responses to calcium-mobilizing hormones in both cell types, but paradoxically reduced overall calcium mobilization. The reduction in calcium mobilization could be attributed to an inhibition of calcium influx in T84 cells, although a different mechanism likely operates in acinar cells. The effects of the cAMP analogue were reproduced by other agents that increase cAMP. Furthermore, econazole, an inhibitor of calcium influx, potentiated secretory responses to calcium-dependent stimulation in T84 cells without itself inducing secretion. We conclude that there is cross-talk between calcium and cAMP-dependent signaling pathways at the level of second messenger generation in two secretory cell types. This cross-talk appears to regulate the extent of secretory responses.


De novo mutations of KIAA2022 in females cause intellectual disability and intractable epilepsy.
Mutations in the KIAA2022 gene have been reported in male patients with X-linked intellectual disability, and related female carriers were unaffected. Here, we report 14 female patients who carry a heterozygous de novo KIAA2022 mutation and share a phenotype characterised by intellectual disability and epilepsy. Reported females were selected for genetic testing because of substantial developmental problems and/or epilepsy. X-inactivation and expression studies were performed when possible. All mutations were predicted to result in a frameshift or premature stop. 12 out of 14 patients had intractable epilepsy with myoclonic and/or absence seizures, and generalised in 11. Thirteen patients had mild to severe intellectual disability. This female phenotype partially overlaps with the reported male phenotype which consists of more severe intellectual disability, microcephaly, growth retardation, facial dysmorphisms and, less frequently, epilepsy. One female patient showed completely skewed X-inactivation, complete absence of RNA expression in blood and a phenotype similar to male patients. In the six other tested patients, X-inactivation was random, confirmed by a non-significant twofold to threefold decrease of RNA expression in blood, consistent with the expected mosaicism between cells expressing mutant or normal KIAA2022 alleles. Heterozygous loss of KIAA2022 expression is a cause of intellectual disability in females. Compared with its hemizygous male counterpart, the heterozygous female disease has less severe intellectual disability, but is more often associated with a severe and intractable myoclonic epilepsy.


A validated and stability indicating HPLC method for analysis of diminazene aceturate and antipyrine combination in a ready injectable solution.
Diminazene aceturate and Antipyrine combination therapy is widely used in veterinary medicine. A simple reverse HPLC method for the analysis of samples of a ready injectable formulation containing a mixture of active ingredients and inactive excipients has been developed. The HPLC analysis was carried out using a reversed phase (RP)-C18 (250 mm×4.0 mm, 5 μm) column. The isocratic mobile phase consisted of a mixture of acetonitrile, methanol, phosphate buffer and hexane sulfonate; the flow rate was 0.6 mL/min and ultraviolet detection was at 291 nm. This method was validated in accordance with FDA and ICH guidelines and showed good linearity, accuracy, precision, selectivity and the system suitability results were within the acceptance criteria. A stability-indicating study was also carried out and indicated that this method could be used for purity and degradation evaluation of these formulations.