FFA2 activation combined with ulcerogenic COX inhibition induces duodenal mucosal injury via the 5-HT pathway in rats.
Serotonin (5-HT), predominantly synthesized and released by enterochromaffin cells, is implicated in gastrointestinal symptoms such as emesis, abdominal pain, and diarrhea. Because luminal short-chain fatty acids (SCFAs) release 5-HT from enterochromaffin cells, which express the SCFA receptor free fatty acid receptor 2 (FFA2) in rat duodenum, we examined the effects of the selective FFA2 agonist phenylacetamide-1 (PA1) on duodenal 5-HT release with consequent bicarbonate secretion [duodenal bicarbonate secretion (DBS)] and on indomethacin (IND)-induced enteropathy. Intestinal injury was induced by IND (10 mg/kg sc) with or without PA1. We measured DBS in vivo in a duodenal loop perfused with PA1 while measuring 5-HT released in the portal vein. Duodenal blood flow was measured by laser-Doppler flowmetry. IND induced small intestinal ulcers with duodenal sparing. PA1 given with IND (IND + PA1) dose dependently induced duodenal erosions. IND + PA1-induced duodenal lesions were inhibited by the FFA2 antagonist GLPG-0974, ondansetron, or omeprazole but not by RS-23597 or atropine. Luminal perfusion of PA1 augmented DBS accompanied by increased portal blood 5-HT concentrations with approximately eight times more release at 0.1 mM than at 1 µM, with the effects inhibited by coperfusion of GLPG-0974. Luminal PA1 at 1 µM increased, but at 0.1 mM diminished, duodenal blood flow. Cosuperfusion of PA1 (0.1 mM) decreased acid-induced hyperemia, further reduced by IND pretreatment but restored by ondansetron. These results suggest that, although FFA2 activation enhances duodenal mucosal defenses, FFA2 overactivation during ulcerogenic cyclooxygenase inhibition may increase the vulnerability of the duodenal mucosa to gastric acid via excessive 5-HT release and 5-HT3 receptor activation, implicated in foregut-related symptoms such as emesis and epigastralgia.NEW & NOTEWORTHY Luminal free fatty acid receptor 2 agonists stimulate enterochromaffin cells and release serotonin, which enhances mucosal defenses in rat duodenum. However, overdriving serotonin release with high luminal concentrations of free fatty acid 2 ligands such as short-chain fatty acids injures the mucosa by decreasing mucosal blood flow. These results are likely implicated in serotonin-related dyspeptic symptom generation because of small intestinal bacterial overgrowth, which is hypothesized to generate excess SCFAs in the foregut, overdriving serotonin release from enterochromaffin cells.


The venous system of E14.5 mouse embryos-reference data and examples for diagnosing malformations in embryos with gene deletions.
Approximately one-third of randomly produced knockout mouse lines produce homozygous offspring, which fail to survive the perinatal period. The majority of these die around or after embryonic day (E)14.5, presumably from cardiovascular insufficiency. For diagnosing structural abnormalities underlying death and diseases and for researching gene function, the phenotype of these individuals has to be analysed. This makes the creation of reference data, which define normal anatomy and normal variations the highest priority. While such data do exist for the heart and arteries, they are still missing for the venous system. Here we provide high-quality descriptive and metric information on the normal anatomy of the venous system of E14.5 embryos. Using high-resolution digital volume data and 3D models from 206 genetically normal embryos, bred on the C57BL/6N background, we present precise descriptive and metric information of the venous system as it presents itself in each of the six developmental stages of E14.5. The resulting data shed new light on the maturation and remodelling of the venous system at transition of embryo to foetal life and provide a reference that can be used for detecting venous abnormalities in mutants. To explore this capacity, we analysed the venous phenotype of embryos from 7 knockout lines (Atp11a, Morc2a, 1700067K01Rik, B9d2, Oaz1, Celf4 and Coro1c). Careful comparisons enabled the diagnosis of not only simple malformations, such as dual inferior vena cava, but also complex and subtle abnormalities, which would have escaped diagnosis in the absence of detailed, stage-specific referenced data.


FLI1 monoallelic expression combined with its hemizygous loss underlies Paris-Trousseau/Jacobsen thrombopenia.
Paris-Trousseau syndrome (PTS; also known as Jacobsen syndrome) is characterized by several congenital anomalies including a dysmegakaryopoiesis with two morphologically distinct populations of megakaryocytes (MKs). PTS patients harbor deletions on the long arm of chromosome 11, including the FLI1 gene, which encodes a transcription factor essential for megakaryopoiesis. We show here that lentivirus-mediated overexpression of FLI1 in patient CD34(+) cells restores the megakaryopoiesis in vitro, indicating that FLI1 hemizygous deletion contributes to the PTS hematopoietic defects. FISH analysis on pre-mRNA and single-cell RT-PCR revealed that FLI1 expression is mainly monoallelic in CD41(+)CD42(-) progenitors, while it is predominantly biallelic in the other stages of megakaryopoiesis. In PTS cells, the hemizygous deletion of FLI1 generates a subpopulation of CD41(+)CD42(-) cells completely lacking FLI1 transcription. We propose that the absence of FLI1 expression in these CD41(+)CD42(-) cells might prevent their differentiation, which could explain the segregation of the PTS MKs into two subpopulations: one normal and one composed of small immature MKs undergoing a massive lysis, presumably originating from either FLI1(+) or FLI1(-) CD41(+)CD42(-) cells, respectively. Thus, we point to the role of transient monoallelic expression of a gene essential for differentiation in the genesis of human haploinsufficiency-associated disease and suggest that such a mechanism may be involved in the pathogenesis of other congenital or acquired genetic diseases.


[Comparison the effects of prilocaine and the addition of dexketoprofen and dexamethasone to prilocaine for intravenous regional anesthesia].
The aim of this study was to compare the anesthetic and analgesic effects of prilocaine alone, prilocaine added dexketoprofen and dexamethasone during intravenous regional anesthesia (IVRA). Forty five patients undergoing forearm or hand surgery were randomly assigned to one of three groups to receive (Group P) 3 mg/kg 0.5% prilocaine; (Group PDK) 3 mg/kg 0.5% prilocaine plus 50 mg dexketoprofen; (Group PDM) 3 mg/kg 0.5% prilocaine plus 8 mg dexamethasone in total 40 ml volume for IVRA. The onset and duration of sensory and motor blocks, hemodynamic datas, duration of analgesia and tourniquet, time to first analgesic requirement, visual analog scale (VAS), total analgesic consumption in 24 hours and patient satisfaction score were assessed and recorded. Time to onset of sensory block was found to be longer in Group P (p<0.05), though no significance was found according to sensory block recovery times amoung groups. Time to onset of motor block was found to be longer and recovery time of motor block was found to be shorter in Group P (p<0.05). Time to first analgesic requirement was found to be longer in Group PDK,and was found to be high in Group PDM than Group PDK(p<0.05). The VAS scores was found to be high and patient satisfaction scale was found to be low in Group P (p<0.05). The addition of dexketoprofen and dexamethasone to prilocaine during IVRA improves the quality of both anesthesia and analgesia moreover dexketoprofen provides beter postoperative analgesia during the first 24 hour after surgery.


Associations of Sleep Disturbance, Atopy, and Other Health Measures with Chronic Overlapping Pain Conditions.
To quantify the contributions of atopic disorders, sleep disturbance, and other health conditions to five common pain conditions. This cross-sectional analysis used data from 655 participants in the OPPERA study. The authors investigated the individual and collective associations of five chronic overlapping pain conditions (COPCs) with medically diagnosed atopic disorders and self-reported sleep disturbance, fatigue, and symptoms of obstructive sleep apnea. Atopic disorders were allergies, allergic rhinitis, atopic dermatitis, allergic asthma, urticaria, allergic conjunctivitis, and food allergy. Logistic regression models estimated odds ratios as measures of association with temporomandibular disorders, headache, irritable bowel syndrome, low back pain, and fibromyalgia. Measures of sleep and atopy disorders were standardized to z scores to determine the relative strength of their associations with each COPC. Sociodemographic characteristics and body mass index were covariates. Random forest regression analyzed all variables simultaneously, computing importance metrics to determine which variables best differentiated pain cases from controls. Fatigue and sleep disturbance were strongly associated with each COPC and with the total number of COPCs. An increase of one standard deviation in fatigue or sleep disturbance score was associated with approximately two-fold greater odds of having a COPC. In random forest models, atopic disorders contributed more than other health measures to differentiating between cases and controls of headache, whereas other COPCs were best differentiated by measures of fatigue or sleep. Atopic disorders, previously recognized as predictors of poor sleep, are associated with COPCs after accounting for sleep problems.


Glutamate preconditioning prevents neuronal death induced by combined oxygen-glucose deprivation in cultured cortical neurons.
The study of ischemic tolerance is critical in the development of strategies for the treatment of ischemic stroke. We used the oxygen and glucose deprivation (OGD) paradigm in cultured cortical neurons as an in vitro approach to elucidate the mechanism of protection conferred by glutamate preconditioning. Pretreatment of neurons with N-methyl-d-aspartate (NMDA) receptor antagonists prevented OGD-induced cell death whereas alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor and voltage-dependent Ca(++) channel (VDCC) blockers were without effect. Neurons preconditioned with glutamate exhibited resistant to damage induced by OGD. The ischemic tolerance depended on the duration of preconditioning exposure and the interval between preconditioning exposure and test challenge. Protective efficacy was blocked by the NMDA or AMPA receptor antagonists but not by the VDCC blocker. Furthermore, neuroprotective effect was not seen if extracellular Ca(++) was omitted or removed with EGTA. Pretreatment with staurosporin and 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN93) but not 2-(4-Morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one (LY294002) or 1,4-diamino-2,3-dicyano-1, 4-bis[2-aminophenylthio] butadiene (U0126) significantly reduced ischemic tolerance. Preconditioning increased phosphorylated levels of cAMP responsive element binding protein (CREB) and pretreatment with CRE-decoy oligonucleotide completely blocked preconditioning-induced increase in cell viability. Importantly, glutamate preconditioning increased Bcl-2 expression that was blocked by KN93, staurosporin and CRE-decoy oligonucleotide. These results suggest that preconditioning with glutamate conferred neuroprotection against subsequent OGD by inducing p-CREB-mediated Bcl-2 expression.


Functional complementation of RNA interference mutants in trypanosomes.
In many eukaryotic cells, double-stranded RNA (dsRNA) triggers RNA interference (RNAi), the specific degradation of RNA of homologous sequence. RNAi is now a major tool for reverse-genetics projects, including large-scale high-throughput screens. Recent reports have questioned the specificity of RNAi, raising problems in interpretation of RNAi-based experiments. Using the protozoan Trypanosoma brucei as a model, we designed a functional complementation assay to ascertain that phenotypic effect(s) observed upon RNAi were due to specific silencing of the targeted gene. This was applied to a cytoskeletal gene encoding the paraflagellar rod protein 2 (TbPFR2), whose product is essential for flagellar motility. We demonstrate the complementation of TbPFR2, silenced via dsRNA targeting its UTRs, through the expression of a tagged RNAi-resistant TbPFR2 encoding a protein that could be immunolocalized in the flagellum. Next, we performed a functional complementation of TbPFR2, silenced via dsRNA targeting its coding sequence, through heterologous expression of the TbPFR2 orthologue gene from Trypanosoma cruzi: the flagellum regained its motility. This work shows that functional complementation experiments can be readily performed in order to ascertain that phenotypic effects observed upon RNAi experiments are indeed due to the specific silencing of the targetted gene. Further, the results described here are of particular interest when reverse genetics studies cannot be easily achieved in organisms not amenable to RNAi. In addition, our strategy should constitute a firm basis to elaborate functional-dissection studies of genes from other organisms.


Characterization of platelet-derived growth factor-A expression in mouse tissues using a lacZ knock-in approach.
Expression of the platelet-derived growth factor A-chain gene (Pdgfa) occurs widely in the developing mouse, where it is mainly localized to various epithelial and neuronal structures. Until now, in situ mRNA hybridization (ISH) has been the only reliable method to identify Pdgfa expression in tissue sections or whole mount preparations. Validated protocols for in situ detection of PDGF-A protein by immunohistochemistry is lacking. In particular, this has hampered understanding of Pdgfa expression pattern in adult tissues, where ISH is technically challenging. Here, we report a gene targeted mouse Pdgfa allele, Pdgfaex4COIN, which is a combined conditional knockout and reporter allele. Cre-mediated inversion of the COIN cassette inactivates Pdgfa coding while simultaneously activating a beta-galactosidase (lacZ) reporter under endogenous Pdgfa transcription control. The generated Pdgfaex4COIN-INV-lacZ allele can next be used to identify cells carrying a Pdgfa null allele, as well as to map endogenous Pdgfa expression. We evaluated the Pdgfaex4COIN-INV-lacZ allele as a reporter for endogenous Pdgfa expression patterns in mouse embryos and adults. We conclude that the expression pattern of Pdgfaex4COIN-INV-lacZ recapitulates known expression patterns of Pdgfa. We also report on novel embryonic and adult Pdgfa expression patterns in the mouse and discuss their implications for Pdgfa physiology.


Mapping and sequencing of structural variation from eight human genomes.
Genetic variation among individual humans occurs on many different scales, ranging from gross alterations in the human karyotype to single nucleotide changes. Here we explore variation on an intermediate scale--particularly insertions, deletions and inversions affecting from a few thousand to a few million base pairs. We employed a clone-based method to interrogate this intermediate structural variation in eight individuals of diverse geographic ancestry. Our analysis provides a comprehensive overview of the normal pattern of structural variation present in these genomes, refining the location of 1,695 structural variants. We find that 50% were seen in more than one individual and that nearly half lay outside regions of the genome previously described as structurally variant. We discover 525 new insertion sequences that are not present in the human reference genome and show that many of these are variable in copy number between individuals. Complete sequencing of 261 structural variants reveals considerable locus complexity and provides insights into the different mutational processes that have shaped the human genome. These data provide the first high-resolution sequence map of human structural variation--a standard for genotyping platforms and a prelude to future individual genome sequencing projects.


Anti-MicroRNA-21 Oligonucleotide Loaded Spermine-Modified Acetalated Dextran Nanoparticles for B1 Receptor-Targeted Gene Therapy and Antiangiogenesis Therapy.
The use of nanoparticles (NPs) to deliver small inhibiting microRNAs (miRNAs) has shown great promise for treating cancer. However, constructing a miRNA delivery system that targets brain cancers, such as glioblastoma multiforme (GBM), remains technically challenging due to the existence of the blood-tumor barrier (BTB). In this work, a novel targeted antisense miRNA-21 oligonucleotide (ATMO-21) delivery system is developed for GBM treatment. Bradykinin ligand agonist-decorated spermine-modified acetalated dextran NPs (SpAcDex NPs) could temporarily open the BTB by activating G-protein-coupled receptors that are expressed in tumor blood vessels and tumor cells, which increase transportation to and accumulation in tumor sites. ATMO-21 achieves high loading in the SpAcDex NPs (over 90%) and undergoes gradual controlled release with the degradation of the NPs in acidic lysosomal compartments. This allows for cell apoptosis and inhibition of the expression of vascular endothelial growth factor by downregulating hypoxia-inducible factor (HIF-1α) protein. An in vivo orthotopic U87MG glioma model confirms that the released ATMO-21 shows significant therapeutic efficacy in inhibiting tumor growth and angiogenesis, demonstrating that agonist-modified SpAcDex NPs represent a promising strategy for GBM treatment combining targeted gene therapy and antiangiogenic therapy.


Transcriptional regulation of flhDC by QseBC and sigma (FliA) in enterohaemorrhagic Escherichia coli.
Enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7, the causative agent of haemorrhagic colitis, has been shown to utilize a cell-to-cell signalling system to regulate gene expression. We have previously reported that the quorum sensing E. coli regulators B and C (QseBC) may act as a two-component system in EHEC to transcriptionally regulate the expression of flagella and motility through flhDC, the master regulator of flagella and motility genes. Here, we performed deletion analyses using the flhDC promoter in order to determine the minimal promoter regions necessary for QseBC transcriptional activation. We also performed electrophoretic mobility shift assays, competition experiments and DNaseI footprints, which suggest that QseB directly binds the flhDC promoter at high- and low-affinity binding sites. These analyses have allowed us to determine the potential consensus sequence to which QseB binds in order to regulate transcription. Additionally, we mapped the transcriptional start site of flhDC responsive to QseBC, leading to the identification of a conserved FliA (sigma28) consensus sequence. These results suggest that FliA (sigma28), a class 2 flagellar gene, may be aiding in the transcriptional initiation of class 1 genes (flhDC) in EHEC. In order to further characterize the role of FliA (sigma28) in transcription of the flhDC promoter, we constructed a fliA isogenic mutant in EHEC. The flhDC::lacZ transcriptional fusion showed decreased activity in the fliA mutant compared with wild-type and complemented strains. Taken together, these results indicate that transcriptional initiation at the flhDC promoter by QseBC appears to be complex and dependent on the presence of FliA (sigma28).


Mesoporous silica particles as potential carriers for protein drug delivery: protein immobilization and the effect of displacer on γ-globulin release.
The adsorption of γ-globulin was evaluated with experiments with silica particles marketed as Syloid AL1-FP (SAL), XDP-3150 (SXDP), and 244FP (SFP). The influence of pH, pore sizes, and degree of surface porosity on the extent of γ-globulin immobilization was examined. Protein adsorption on these particles was largely related to their surface porosity and pore sizes. The adsorption capacity was established to be greater with mesoporous SFP and SXDP particles at 474 and 377 mg/g, respectively, when compared to significantly low-porosity SAL (16 mg/g). Additionally, γ-globulin immobilization was favored at pH closer to iso-electric point. A key aim of this work was to better understand and improve the limited reversibility of protein adsorption. Protein desorption was found to be lower in simulated intestinal fluid (SIF) in comparison to pH 7.4 phosphate buffer (PB). The use of displacer molecules (sodium dodecyl sulfate [SDS]/Tween 80/Pluronic F127 [PF127]) promoted protein desorption from the adsorbent surface by the exchange mechanism. The PF127 provided substantial release in both studied condition but the highest release of 83% of γ-globulin from SXDP was obtained with tween 80 in PB. The released protein was analyzed with circular dichroism (CD) spectroscopy which indicated that the secondary structure of desorbed γ-globulin was dependent on the pH and displacer molecule. The conformation largely remained unchanged when desorption was carried out in SIF but changed markedly in PB specially in the presence of SDS.


Specificity and mutational analysis of the metal-dependent 3-deoxy-D-manno-octulosonate 8-phosphate synthase from Acidithiobacillus ferrooxidans.
3-deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the reaction between phosphoenol pyruvate and D-arabinose 5-phosphate to generate KDO8P. This reaction is part of the biosynthetic pathway to 3-deoxy-D-manno-octulosonate, a component of the lipopolysaccharide of the Gram-negative bacterial cell wall. Two distinct groups of KDO8PSs exist, differing by the absolute requirement of a divalent metal ion. In this study Acidithiobacillus ferrooxidans KDO8PS has been expressed and purified and shown to require a divalent metal ion, with Mn2+, Co2+ and Cd2+ (in decreasing order) being able to restore activity to metal-free enzyme. Cd2+ significantly enhanced the stability of the enzyme, raising the Tm by 14 degrees C. D-glucose 6-phosphate and D-erythrose 4-phosphate were not substrates for A. ferrooxidans KDO8PS, whereas 2-deoxy-D-ribose 5-phosphate was a poor substrate and there was negligible activity with D-ribose 5-phosphate. The 243AspGlyPro245 motif is absolutely conserved in the metal-independent group of synthases, but the Gly and Pro sites are variable in the metal-dependent enzymes. Substitution of the putative metal-binding Asp243 to Ala in A. ferrooxidans KDO8PS gave inactive enzyme, whereas substitutions Asp243Glu or Pro245Ala produced active enzymes with altered metal-dependency profiles. Prior studies indicated that exchange of a metal-binding Cys for Asn converts metal-dependent KDO8P synthase into a metal-independent form. Unexpectedly, this mutation in A. ferrooxidans KDO8P synthase (Cys21Asn) gave inactive enzyme. This finding, together with modest activity towards 2-deoxy-D-ribose 5-phosphate suggests similarities between the A. ferrooxidans KDO8PS and the related metal-dependent 3-deoxy-D-arabino-heptulosonate phosphate synthase, and highlights the importance of the AspGlyPro loop in positioning the substrate for effective catalysis in all KDO8P synthases.


Effect of intrathecal oxcarbazepine on rat tail flick test-determined morphine tolerance.
Repeated administration of morphine leads to characteristic tolerance. We tested the effects of intrathecal oxcarbazepine (OXC) on spinal morphine tolerance in rats using the tail flick test. Sprague-Dawley rats received intrathecal injections of 10 microliter saline alone, or 10 microliter of solutions containing 100 microgram OXC, 15 microgram morphine, or OXC + morphine for 7 days. Different groups of rats received OXC on days 1-7, 1-3, or 5-7. The tail-flick assay was used to measure acute and chronic nociception. The nociceptive stimulus consisted of dipping the distal 5 cm of the tail into warm water before and 30 min after drug injection. On day 8, an antinociceptive dose-response curve was plotted, and the 50% effective dose for morphine (given alone) was determined for all groups. Morphine or OXC both produced acute antinociception; OXC + morphine resulted in a significantly larger response than obtained with morphine alone. Morphine tolerance was produced by intrathecal injection of morphine over 7 days. Co-administration of morphine and OXC completely blocked morphine tolerance, but tolerance developed when OXC injection was stopped, and morphine potency was partially restored by co-administration of OXC in tolerant rats. The antinociceptive effect of both acute and chronic morphine therapy is increased with intrathecal OXC, and antinociceptive morphine tolerance is attenuated in rats.


Cervical Ectopic Pregnancy Complicated by Escherichia Coli Septicemia: A Case Report.
Cervical ectopic pregnancy is an uncommon form of ectopic pregnancy. The spectrum of treatment options includes systemic medical therapy, local injection with methotrexate and/or potassium chloride, surgery, or a combination of these modalities. A 29-year-old woman, gravida 3, aborta 2, was found to have a cervical ectopic pregnancy on ultrasound at 5+6 weeks' gestation. She presented to the hospital with vaginal bleeding. Treatment was initiated with multidose methotrexate and leucovorin rescue. She subsequently developed Gram-negative septicemia, with blood cultures growing Escherichia coli. This was managed successfully by surgical removal of the ectopic pregnancy and antimicrobial therapy. A cervical ectopic pregnancy can be complicated by E. coli septicemia.


Novel superhard carbon: C-centered orthorhombic C8.
A novel carbon allotrope of C-centered orthorhombic C(8) (Cco-C(8)) is predicted by using a recently developed particle-swarm optimization method on structural search. Cco-C(8) adopts a sp(3) three-dimensional bonding network that can be viewed as interconnected (2,2) carbon nanotubes through 4- and 6-member rings and is energetically more favorable than earlier proposed carbon polymorphs (e.g., M carbon, bct-C(4), W carbon, and chiral C(6)) over a wide range of pressures studied (0-100 GPa). The simulated x-ray diffraction pattern, density, and bulk modulus of Cco-C(8) are in good accordance with the experimental data on structurally undetermined superhard carbon recovered from cold compression of carbon nanotube bundles. The simulated hardness of Cco-C(8) can reach a remarkably high value of 95.1 GPa, such that it is capable of cracking diamond.


Phosphate metabolism in red blood cells of critically ill neonates.
Very few data exist on phosphate metabolism in critically ill neonates. Therefore we studied the incidence of hypophosphataemia, the intracellular metabolism of phosphate by measuring adenosine 5'-triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) in red blood cells, and excretion of phosphate in urine. The aims of the study were early detection of changes in phosphate metabolism as possible diagnostic markers of sepsis and defining the cause of hypophosphataemia. Neonates, treated in multidisciplinary paediatric intensive care unit (PICU), included in the study, were less than three days of age. Eighteen of them had respiratory distress syndrome (RDS) and 16 had microbiologically confirmed or clinical sepsis. The overall incidence of hypophosphataemia in critically ill neonates was over 80%, and was more common (88%) and more profound in those with sepsis than in those with RDS (79%). Therefore the septic neonates needed significantly larger amounts of phosphate to maintain normophosphataemia. In septic neonates ATP concentration in red blood cells was significantly lower than in neonates with RDS and controls, while the 2,3-DPG concentration was increased as a result of compensation. In septic neonates urinary losses of inorganic phosphate (Pi) were significantly higher than in neonates with RDS. Hypophosphataemia in critically ill neonates is at least partly due to higher urinary losses of phosphate.


Intracellular immunization of rhesus CD34+ hematopoietic progenitor cells with a hairpin ribozyme protects T cells and macrophages from simian immunodeficiency virus infection.
Evaluation of candidate genes for stem cell gene therapy for acquired immunodeficiency syndrome (AIDS) has been limited by the difficulty of supporting in vitro T-cell differentiation of genetically modified hematopoietic progenitor cells. Using a novel thymic stromal culture technique, we evaluated the ability of a hairpin ribozyme specific for simian immunodeficiency virus (SIV) and human immunodeficiency virus type 2 (HIV-2) to inhibit viral replication in T lymphocytes derived from transduced CD34+ progenitor cells. Retroviral transduction of rhesus macaque CD34+ progenitor cells with a retroviral vector (p9456t) encoding the SIV-specific ribozyme and the selectable marker neomycin phosphotransferase in the presence of bone marrow stroma and in the absence of exogenous cytokines resulted in efficient transduction of both colony-forming units and long-term culture-initiating cells, with transduction efficiencies ranging between 21% and 56%. After transduction, CD34+ cells were cultured on rhesus thymic stromal culture (to support in vitro differentiation of T cells) or in the presence of cytokines (to support differentiation of macrophage-like cells). After expansion and selection with the neomycin analog G418, cells derived from transduced progenitor cells were challenged with SIV. CD4+ T cells derived from CD34+ hematopoietic cells transduced with the ribozyme vector p9456t were highly resistant to challenge with SIV, exhibiting up to a 500-fold decrease in SIV replication, even after high multiplicities of infection. Macrophages derived from CD34+ cells transduced with the 9456 ribozyme exhibited a comparable level of inhibition of SIV replication. These results show that a hairpin ribozyme introduced into CD34+ hematopoietic progenitor cells can retain the ability to inhibit AIDS virus replication after T-cell differentiation and support the feasibility of intracellular immunization of hematopoietic stem cells against infection with HIV and SIV. Protection of multiple hematopoietic lineages with the SIV-specific ribozyme should permit analysis of stem cell gene therapy for AIDS in the SIV/macaque model.


Efficacy of antimicrobial-impregnated external ventricular drain catheters: a prospective, randomized, controlled trial.
Catheter-related infection of the cerebrospinal fluid (CSF) pathways is a potentially life-threatening complication of external ventricular drainage. A major source of infection is bacterial contamination along the external ventricular drain (EVD) catheter track. The authors examined the efficacy of EVD catheters impregnated with minocycline and rifampin in preventing these catheter-related infections. The authors conducted a prospective, randomized clinical trial at six academic medical centers. All hospitalized patients 18 years or older who required placement of an EVD catheter were eligible for inclusion in the study. Patients were randomly assigned to undergo placement of an EVD with a catheter impregnated with minocycline and rifampin or a standard untreated catheter (control group). To assess primary outcome, CSF samples were collected using a sterile technique at the time of catheter insertion, at least every 72 hours while the catheter remained in place, and at the time of catheter removal. At the time of removal, CSF cultures were obtained from the tip and tunneled segments of each catheter by performing semiquantitative roll-plate and quantitative sonication techniques. Of the 306 patients enrolled in the study, data from 288 were included in the final analysis. Eighteen patients were excluded from analysis: 14 because the ventricular catheter was in place less than 24 hours, and four because CSF cultures obtained at the time of catheter insertion were positive for infection. Of these 288 patients, 139 were assigned to the control group and 149 to the treatment group. The two groups were well matched with respect to all clinical characteristics, including patient sex and mean age, indication for catheter placement, and length of time the catheter remained in place. The antibiotic-impregnated catheters were one half as likely to become colonized as the control catheters (17.9 compared with 36.7%, respectively, p < 0.0012). Positive CSF cultures were seven times less frequent in patients with antibiotic-impregnated catheters compared with those in the control group (1.3 compared with 9.4%, respectively, p = 0.002). The use of EVD catheters impregnated with minocycline and rifampin can significantly reduce the risk of catheter-related infections.


HLA Upregulation During Dengue Virus Infection Suppresses the Natural Killer Cell Response.
Dengue virus (DENV) is the most prevalent mosquito-borne virus in the world and a major cause of morbidity in the tropics and subtropics. Upregulation of HLA class I molecules has long been considered a feature of DENV infection, yet this has not been evaluated in the setting of natural infection. Natural killer (NK) cells, an innate immune cell subset critical for mounting an early response to viral infection, are inhibited by self HLA class I, suggesting that upregulation of HLA class I during DENV infection could dampen the NK cell response. Here we addressed whether upregulation of HLA class I molecules occurs during in vivo DENV infection and, if so, whether this suppresses the NK cell response. We found that HLA class I expression was indeed upregulated during acute DENV infection across multiple cell lineages in vivo. To better understand the role of HLA class I upregulation, we infected primary human monocytes, a major target of DENV infection, in vitro. Upregulation of total HLA class I is dependent on active viral replication and is mediated in part by cytokines and other soluble factors induced by infection, while upregulation of HLA-E occurs in the presence of replication-incompetent virus. Importantly, blocking DENV-infected monocytes with a pan-HLA class I Fab nearly doubles the frequency of degranulating NK cells, while blocking HLA-E does not significantly improve the NK cell response. These findings demonstrate that upregulation of HLA class I during DENV infection suppresses the NK cell response, potentially contributing to disease pathogenesis.


Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells.
Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.


Research and organizational issues for the implementation of family work in community psychiatric services.
The need for evidence-based practice (EBP) to guide and develop mental health services remains fundamental for modern services. Aim. To discuss issues that impact upon implementation of EBP and practice development using family work (FW) as an example. A selection of the FW literature was reviewed drawing on sources including the Cochrane Library, Cinahl and Medline. Keywords used were FW, community mental health team and research design. Centralized policy initiatives and guidelines that are themselves guided by evidence of randomized controlled trials predominantly risk alienating practitioners and clients/carers. Family work has some demonstrable clinical benefits although models differ and the active therapeutic agent remains unclear. Its adoption into routine care is also hindered by a productivity management outlook that seeks to maximize stretched resources and whose values are likely to be internalized by practitioners. The dichotomous position of previous research and practice development make implementation of EBP difficult and highlights the need for strategic planning that embraces both factors. The current drive to increase EBP requires a bi-directional process of influence that allows individual practitioners and clients/carers to become producers of evidence and not simply recipients. The authors support wider adoption of case study research designs to reflect the unpredictable nature of mental health care. Adoption of assertive community treatment models within community services is most likely to promote the excellence management model and accommodate EBP such as FW.


Reverse microdialysis of a dopamine uptake inhibitor in the nucleus accumbens of alcohol-preferring rats: effects on dialysate dopamine levels and ethanol intake.
The mesolimbic dopamine (DA) system has been implicated in mediating the reinforcing actions of ethanol (EtOH). This study examines the effects of local perfusion of the DA uptake inhibitor GBR12909 (GBR) on (1) DA levels in the nucleus accumbens (NAc) and (2) EtOH drinking in alcohol-preferring rats. Stable drinking of a 15% (v/v) EtOH solution (minimum of 0.75 g/kg body weight) was established in daily 1 hr limited access sessions. Rats were then implanted with bilateral guide cannulae aimed 4 mm above the NAc. After recovery from surgery, concentric microdialysis probes (2 mm dialysis membrane surface) were inserted into the NAc. Most placements were in the shell or overlapping both shell and core. Two days later, the probes were perfused at 1.0 microl/min with artificial cerebral spinal fluid (aCSF) for at least a 90 min washout period followed by collection of five basal samples over 150 min. Rats were then perfused with either aCSF alone or 10, 25, 100, or 200 microM of GBR for 240 min on the first day of microdialysis. During the last 60 min of the drug treatment phase, rats were given their scheduled access to 15% EtOH. All rats were then perfused with aCSF for the last 90 min of the experiment. The following day, the procedure was repeated, but animals that received aCSF on the first day were given a dose of GBR and rats given GBR on the first day received only aCSF. GBR perfusion increased extracellular NAc DA levels dose dependently to more than 800% of basal levels at 100 to 200 microM but failed to alter EtOH intake (p > 0.05, paired t test) at any concentration tested. Moreover, after 100 microM of GBR perfusion had terminated, the extracellular levels of DA in the NAc remained elevated for approximately 24 hr (790% of day 1 basal; p < 0.05). The increase in dialysate DA levels observed during GBR perfusion with 100 microM was significantly greater for EtOH-experienced rats than for EtOH-naïve rats [F(7,59) = 14.85, p < 0.0001, analysis of variance, Student-Newman-Keuls post hoc test]. The results suggest (1) that EtOH drinking experience induces neuroadaptations that increase DA release in the NAc, and (2) that additional elevation in synaptic levels of DA in the NAc does not influence the maintenance of ongoing alcohol drinking under scheduled access conditions in alcohol-preferring animals.


Mitochondrial Respiration Is Reduced in Atherosclerosis, Promoting Necrotic Core Formation and Reducing Relative Fibrous Cap Thickness.
Mitochondrial DNA (mtDNA) damage is present in murine and human atherosclerotic plaques. However, whether endogenous levels of mtDNA damage are sufficient to cause mitochondrial dysfunction and whether decreasing mtDNA damage and improving mitochondrial respiration affects plaque burden or composition are unclear. We examined mitochondrial respiration in human atherosclerotic plaques and whether augmenting mitochondrial respiration affects atherogenesis. Human atherosclerotic plaques showed marked mitochondrial dysfunction, manifested as reduced mtDNA copy number and oxygen consumption rate in fibrous cap and core regions. Vascular smooth muscle cells derived from plaques showed impaired mitochondrial respiration, reduced complex I expression, and increased mitophagy, which was induced by oxidized low-density lipoprotein. Apolipoprotein E-deficient (ApoE-/-) mice showed decreased mtDNA integrity and mitochondrial respiration, associated with increased mitochondrial reactive oxygen species. To determine whether alleviating mtDNA damage and increasing mitochondrial respiration affects atherogenesis, we studied ApoE-/- mice overexpressing the mitochondrial helicase Twinkle (Tw+/ApoE-/-). Tw+/ApoE-/- mice showed increased mtDNA integrity, copy number, respiratory complex abundance, and respiration. Tw+/ApoE-/- mice had decreased necrotic core and increased fibrous cap areas, and Tw+/ApoE-/- bone marrow transplantation also reduced core areas. Twinkle increased vascular smooth muscle cell mtDNA integrity and respiration. Twinkle also promoted vascular smooth muscle cell proliferation and protected both vascular smooth muscle cells and macrophages from oxidative stress-induced apoptosis. Endogenous mtDNA damage in mouse and human atherosclerosis is associated with significantly reduced mitochondrial respiration. Reducing mtDNA damage and increasing mitochondrial respiration decrease necrotic core and increase fibrous cap areas independently of changes in reactive oxygen species and may be a promising therapeutic strategy in atherosclerosis.


[The effect of the carbon sources and calcium ions on the activity of thermostable alpha-amylase in Bacillus sp. 86].
Amylolytic activity of Bacillus sp. 86 thermophilic strain was studied as affected by carbon sources. Glucose and saccharose being added to the nutrition medium with baker's yeast as the basic source of carbon the activity of alpha-amylase decreased. Introduction of native or soluble starch to the medium promoted a sharp increase in the enzymic activity. Bacillus sp. 86 being periodically cultivated in the optimized medium containing 4% of soluble starch and 0.5% of CaCl2, an eight-fold increase of amylolytic activity of cultural liquid was achieved as compared to the enzyme activity in the primary medium.


Immunomodulatory effects of helminths and protozoa in multiple sclerosis and experimental autoimmune encephalomyelitis.
Multiple sclerosis is a chronic inflammatory CNS disease, which affects about 1 in 1000 individuals in the western world. During the last couple of decades, epidemiological data have accumulated, pointing towards increases in incidence. This has been suggested to be linked to the relatively high hygiene standards that exist in the western world, with reduced exposure to various pathogens, including parasites, as a consequence. Parasites are known to employ various immunomodulatory and anti-inflammatory strategies, which enable them to evade destruction by the immune system. This is most likely one of the reasons for the disease-dampening effects, reported in numerous studies investigating parasite infections and autoimmunity. This review will focus on recent advances in the field of parasites as beneficial immunomodulators, in multiple sclerosis and the animal model experimental autoimmune encephalomyelitis.


Partially folded structure of monomeric bovine beta-lactoglobulin.
Bovine beta-LG (beta-lactoglobulin) has been studied under a variety of solution conditions by one- and two-dimensional NMR spectroscopy. At highly acidic pH (pH=2) and low ionic strength the protein is present in a monomeric form, exhibiting a highly structured beta-sheet core and less ordered regions as evidenced by both CD data and the NOESY spectra. Marginal protection was observed for most of the amide protons as a result of high conformational mobility. This structural state of beta-LG may be considered as an attractive model for a partially folded structure occurring late in the folding process of the protein.


A phenylalanine transfer RNA azo dye complex in livers of rats fed diets containing 3'-methyl-4-dimethylaminoazobenzene.
A highly purified phenylalanine transfer RNA (tRNAPhe) was isolated from a normal rat liver and from livers of male and female rats fed a semisynthetic diet containing 0.06% 3'-methyl-4-dimethylaminoazobenzene for 3 weeks. Absorption spectral analysis on tRNAPhe from normal and carcinogen-fed rats indicated an unusual absorption above 335 nm by tRNAPhe isolated from the latter group. Ribonuclease T1 digestion followed by chromatography on diethylaminoethyl cellulose columns indicated the existence of a major peak of covalently bound oligonucleotide-azo complex. Microcrystalline cellulose thin-layer chromatography resolved the peak into one major and four minor components, all with similar absorption spectra. Enzymatic digestion of the major peak obtained from diethylaminoethyl cellulose chromatography to ribonucleosides, followed by chromatography on cellulose thin layer, resulted in one major and three minor components. A higher uridine:cytidine base ratio was also observed in the tRNAPhe isolated from the carcinogenfed animals. These findings suggest that certain transfer RNA's may be major targets for this azo carcinogen.


Genetic Parameters for Reproductive Traits in the Italian Mediterranean Buffalo Using Milk Yield as correlated trait.
Until now, the genetic evaluation of the Italian Mediterranean Buffalo has been mainly focused on production traits. However, female fertility affects the efficiency of the dairy industry as it is essential to maintain the profitability of dairy farms. Indeed, the estimation of its genetic component is crucial for its improvement. In this study, 3 measures of buffalo's fertility were analyzed: the age at first calving (AFC), the interval between first and second calving (CI1), and the interval between second and successive calvings (CI2_12). Milk yield at 270 d (MY270) was used as a correlated trait. First, genetic parameters were estimated using 7,915 buffalo cows with first calving from 1991 to 2018, then breeding values were calculated from 236,087 buffalo cows. Genetic parameters were estimated by Bayesian inference fitting a multiple-trait animal model using the GIBBS1F90 program while BLUPF90 was used for estimation of breeding value. The heritability and repeatability estimates of fertility traits were low. The genetic correlations among fertility traits ranged from 0.10 (AFC-CI1) to 0.92 (CI1-CI2_12). Genetic correlation between MY270 and fertility traits was unfavorable, ranging from 0.23 to 0.48. The results from this study can be used as a basis for the future genetic improvement of fertility traits in the Italian Mediterranean Buffaloes.


Naturopathic Management of Urinary Tract Infections: A Retrospective Chart Review.
Introduction: Antibiotic overuse is a significant driver of bacterial resistance. Urinary tract infections (UTIs, cystitis) are the most common condition for which antibiotics are prescribed in the ambulatory setting. Many complementary and integrative approaches to cystitis have been proposed, including probiotics, D-mannose, and several herbal therapies. Trials comparing such therapies with placebo or antibiotics showed mixed, but promising, results. Naturopathy is a system of medicine that has potential to avoid antibiotic use for UTI because of its affinity for nonpharmacologic therapies and its theory that infection is a result of both the immune system's vulnerability and the pathogen's virulence. Methods: The authors conducted a retrospective chart review of cases treated at four naturopathic clinics in the Portland, OR, metro area, where naturopathic doctors (NDs) have a scope of practice consistent with their license as primary care providers. The primary aim was to characterize how NDs treat UTIs in a real-world setting. Secondary aims were to gather preliminary evidence on the types of patient cases receiving such treatments, outcomes of treatments, and associations between presentation and treatment prescriptions. Results: The authors found 82 distinct treatment regimens among 103 individual patients diagnosed with UTI. Most patients received a combination of herbal medicine and behavioral modification (e.g., increase fluid intake), whereas the most common monotherapeutic regimen was antibiotics. Of the 43 patients who were followed up, 15 had no success with nonpharmacologic therapies and required antibiotics. The sample was comparable with national data regarding composition of public versus private insurance, acute versus recurrent/chronic UTI, and percent of cases related to uropathogenic Escherichia coli. Conclusions: NDs practicing in a primary care context frequently prescribe antibiotic and nonantibiotic multimodal therapy for uncomplicated UTI. These results may guide future studies testing complementary and integrative therapies for uncomplicated UTI.


Antiretroviral drug resistance testing in adult HIV-1 infection: 2008 recommendations of an International AIDS Society-USA panel.
Resistance to antiretroviral drugs remains an important limitation to successful human immunodeficiency virus type 1 (HIV-1) therapy. Resistance testing can improve treatment outcomes for infected individuals. The availability of new drugs from various classes, standardization of resistance assays, and the development of viral tropism tests necessitate new guidelines for resistance testing. The International AIDS Society-USA convened a panel of physicians and scientists with expertise in drug-resistant HIV-1, drug management, and patient care to review recently published data and presentations at scientific conferences and to provide updated recommendations. Whenever possible, resistance testing is recommended at the time of HIV infection diagnosis as part of the initial comprehensive patient assessment, as well as in all cases of virologic failure. Tropism testing is recommended whenever the use of chemokine receptor 5 antagonists is contemplated. As the roll out of antiretroviral therapy continues in developing countries, drug resistance monitoring for both subtype B and non-subtype B strains of HIV will become increasingly important.


The ligand binding mechanism to purine nucleoside phosphorylase elucidated via molecular dynamics and machine learning.
The study of biomolecular interactions between a drug and its biological target is of paramount importance for the design of novel bioactive compounds. In this paper, we report on the use of molecular dynamics (MD) simulations and machine learning to study the binding mechanism of a transition state analogue (DADMe-immucillin-H) to the purine nucleoside phosphorylase (PNP) enzyme. Microsecond-long MD simulations allow us to observe several binding events, following different dynamical routes and reaching diverse binding configurations. These simulations are used to estimate kinetic and thermodynamic quantities, such as kon and binding free energy, obtaining a good agreement with available experimental data. In addition, we advance a hypothesis for the slow-onset inhibition mechanism of DADMe-immucillin-H against PNP. Combining extensive MD simulations with machine learning algorithms could therefore be a fruitful approach for capturing key aspects of drug-target recognition and binding.


Seizure forecasting using machine learning models trained by seizure diaries.
Objectives.People with refractory epilepsy are overwhelmed by the uncertainty of their next seizures. Accurate prediction of future seizures could greatly improve the quality of life for these patients. New evidence suggests that seizure occurrences can have cyclical patterns for some patients. Even though these cyclicalities are not intuitive, they can be identified by machine learning (ML), to identify patients with predictable vs unpredictable seizure patterns.Approach.Self-reported seizure logs of 153 patients from the Human Epilepsy Project with more than three reported seizures (totaling 8337 seizures) were used to obtain inter-seizure interval time-series for training and evaluation of the forecasting models. Two classes of prediction methods were studied: (1) statistical approaches using Bayesian fusion of population-wise and individual-wise seizure patterns; and (2) ML-based algorithms including least squares, least absolute shrinkage and selection operator, support vector machine (SVM) regression, and long short-term memory regression. Leave-one-person-out cross-validation was used for training and evaluation, by training on seizure diaries of all except one subject and testing on the left-out subject.Main results.The leading forecasting models were the SVM regression and a statistical model that combined the median of population-wise seizure time-intervals with a test subject's prior seizure intervals. SVM was able to forecast 50%, 70%, 81%, 84%, and 87% of seizures of unseen subjects within 0, 1, 2, 3 to 4 d of mean absolute forecasting error, respectively. The subject-wise performances show that patients with more frequent seizures were generally better predicted.Significance.ML models can leverage non-random patterns within self-reported seizure diaries to forecast future seizures. While diary-based seizure forecasting alone is only one of many aspects of clinical care of patients with epilepsy, studying the level of predictability across seizures and patients paves the path towards a better understanding of predictable vs unpredictable seizures on individualized and population-wise bases.


Photobleaching of melanosomes from retinal pigment epithelium: II. Effects on the response of living cells to photic stress.
Melanosomes of the retinal pigment epithelium (RPE) are long lived organelles that may undergo photobleaching with aging, which can diminish the antioxidant efficiency of melanin. Here, isolated porcine RPE melanosomes were experimentally photobleached with visible light to simulate aging and compared with untreated granules or control particles (black latex beads) for their effects on the survival of photically stressed ARPE-19 cultures. Particles were delivered to cultures for uptake by phagocytosis then cells were exposed to violet light and analyzed by a new live cell imaging method to identify the time of apoptotic blebbing as a dynamic measure of reduced cell survival. Results indicated that untreated melanosomes did not decrease photic injury to ARPE-19 cells when compared with cells lacking particles or with cells containing control particles, as might be expected if melanin performed an antioxidant function. Instead cells with untreated melanosomes showed reduced survival indicated by an earlier onset of blebbing and a lower fraction of surviving cells after photic stress. Cell survival was reduced even further in stressed cells containing melanosomes that were photobleached, and survival decreased with increasing photobleaching time. Photobleaching of RPE melanosomes therefore makes cells containing them more sensitive to light-induced cytotoxicity. This observation raises the possibility that aged melanosomes increase RPE cell photic stress in situ, perhaps contributing to reduced tissue function and to degeneration of the adjacent retina that the RPE supports. How melanosomes (photobleached or not) interact with their local subcellular environment to modify RPE cell survival is poorly understood and is likely determined by the physicochemical state of the granule and its constituent melanin. The live cell imaging method introduced here, which permitted detection of a graded effect of photobleaching, provides a sensitive bioassay for probing the effects of melanosome modifications.


p53 Binds Preferentially to Non-B DNA Structures Formed by the Pyrimidine-Rich Strands of GAA·TTC Trinucleotide Repeats Associated with Friedreich's Ataxia.
Expansions of trinucleotide repeats (TNRs) are associated with genetic disorders such as Friedreich's ataxia. The tumor suppressor p53 is a central regulator of cell fate in response to different types of insults. Sequence and structure-selective modes of DNA recognition are among the main attributes of p53 protein. The focus of this work was analysis of the p53 structure-selective recognition of TNRs associated with human neurodegenerative diseases. Here, we studied binding of full length p53 and several deletion variants to TNRs folded into DNA hairpins or loops. We demonstrate that p53 binds to all studied non-B DNA structures, with a preference for non-B DNA structures formed by pyrimidine (Py) rich strands. Using deletion mutants, we determined the C-terminal DNA binding domain of p53 to be crucial for recognition of such non-B DNA structures. We also observed that p53 in vitro prefers binding to the Py-rich strand over the purine (Pu) rich strand in non-B DNA substrates formed by sequence derived from the first intron of the frataxin gene. The binding of p53 to this region was confirmed using chromatin immunoprecipitation in human Friedreich's ataxia fibroblast and adenocarcinoma cells. Altogether these observations provide further evidence that p53 binds to TNRs' non-B DNA structures.


IMGT, the international ImMunoGeneTics information system, http://imgt.cines.fr: the reference in immunoinformatics.
IMGT, the international ImMunoGeneTics information system (http://imgt.cines.fr), is a high quality integrated information system specializing in immunoglobulins (IG), T cell receptors (TR), major histocompatibility complex (MHC) and related proteins of the immune system of human and other vertebrates, created in 1989, by the Laboratoire d'ImmunoGénétique Moléculaire (LIGM), at the Université Montpellier II, CNRS, Montpellier, France. IMGT is the global reference in immunogenetics and immunoinformatics and provides a common access to standardized data which include nucleotide and protein sequences, oligonucleotide primers, gene maps, genetic polymorphisms, specificities, 2D and 3D structures. IMGT includes three sequence databases (IMGT/LIGM-DB, IMGT/MHC-DB hosted at EBI, IMGT/PRIMER-DB), one genome database (IMGT/GENE-DB), one 3D structure database (IMGT/3Dstructure-DB), Web resources comprising 8000 HTML pages ("IMGT Marie-Paule page") and interactive tools for sequence (IMGT/V-QUEST, IMGT/JunctionAnalysis, IMGT/Allele-Align, IMGT/PhyloGene) and genome (IMGT/GeneSearch, IMGT/GeneView, IMGT/LocusView) analysis. IMGT data are expertly annotated according to the rules of the IMGT Scientific chart, based on the IMGT-ONTOLOGY concepts. IMGT tools are particularly useful for the analysis of the IG and TR repertoires in physiological normal and pathological situations. IMGT has important applications in medical research (repertoire analysis in autoimmune diseases, AIDS, leukemias, lymphomas, myelomas), biotechnology related to antibody engineering (phage displays, combinatorial libraries) and therapeutic approaches (graft, immunotherapy). IMGT is freely available at http://imgt.cines.fr.


Neuromuscular adaptations to different modes of combined strength and endurance training.
The present study investigated neuromuscular adaptations between same-session combined strength and endurance training with 2 loading orders and different day combined training over 24 weeks. 56 subjects were divided into different day (DD) combined strength and endurance training (4-6 d·wk(-1)) and same-session combined training: endurance preceding strength (E+S) or vice versa (S+E) (2-3 d·wk(-1)). Dynamic and isometric strength, EMG, voluntary activation, muscle cross-sectional area and endurance performance were measured. All groups increased dynamic one-repetition maximum (p<0.001; DD 13±7%, E+S 12±9% and S+E 17±12%) and isometric force (p<0.05-0.01), muscle cross-sectional area (p<0.001) and maximal power output during cycling (p<0.001). DD and S+E increased voluntary activation during training (p<0.05-0.01). In E+S no increase in voluntary activation was detected after 12 or 24 weeks. E+S also showed unchanged and S+E increased maximum EMG after 24 weeks during maximal isometric muscle actions. A high correlation (p<0.001, r=0.83) between the individual changes in voluntary activation and maximal knee extension force was found for E+S during weeks 13-24. Neural adaptations showed indications of being compromised and highly individual relating to changes in isometric strength when E+S-training was performed, while gains in one-repetition maximum, endurance performance and hypertrophy did not differ between the training modes.


Risk factors for tuberculosis in older children and adolescents: a matched case-control study in Recife, Brazil.
Tuberculosis is a major disease worldwide and most research focus on risk factors for adults, although there is a marked adolescent peak in incidence. The objective of this study was to identify risk factors for tuberculosis in children aged 7 to 19. A case control study matched by age with 169 cases and 477 controls. The study population consisted of adolescents and older children from Recife, Brazil. Cases were individuals diagnosed with tuberculosis in the control programme and controls were selected in the neighborhood of cases. Conditional logistic regression was used to identify risk factors. Cigarette smoking increased by 50% the risk of tuberculosis but that this was not statistically significant (OR = 1.6). Other risk factors were sleeping in the same house as a case of tuberculosis (OR = 31.6), living in a house with no piped water (OR = 7.7) (probably as a proxy for bad living conditions), illiteracy (OR = 3.7) and male sex (OR = 1.8). The increase in risk with living in houses with no piped water was much more marked in males. The proportion of cases of tuberculosis attributed to contact with someone with TB was 38% and to illiteracy, lack of piped water and smoking, 20%. Household contact with tuberculosis, social factors and male sex play the biggest role in determining risk of TB disease among children and adolescents in the study. We recommend further research on the relationship of cigarette smoking on tuberculosis in adolescents, and on whether the sex differentials are more marked in bad living conditions. Separate studies should be conducted in older children and in adolescents.


Telomeres and centromeres have interchangeable roles in promoting meiotic spindle formation.
Telomeres and centromeres have traditionally been considered to perform distinct roles. During meiotic prophase, in a conserved chromosomal configuration called the bouquet, telomeres gather to the nuclear membrane (NM), often near centrosomes. We found previously that upon disruption of the fission yeast bouquet, centrosomes failed to insert into the NM at meiosis I and nucleate bipolar spindles. Hence, the trans-NM association of telomeres with centrosomes during prophase is crucial for efficient spindle formation. Nonetheless, in approximately half of bouquet-deficient meiocytes, spindles form properly. Here, we show that bouquet-deficient cells can successfully undergo meiosis using centromere-centrosome contact instead of telomere-centrosome contact to generate spindle formation. Accordingly, forced association between centromeres and centrosomes fully rescued the spindle defects incurred by bouquet disruption. Telomeres and centromeres both stimulate focal accumulation of the SUN domain protein Sad1 beneath the centrosome, suggesting a molecular underpinning for their shared spindle-generating ability. Our observations demonstrate an unanticipated level of interchangeability between the two most prominent chromosomal landmarks.


New criteria for the omission of lymphadenectomy in endometrioid carcinoma.
To establish new criteria for the omission of lymphadenectomy in patients with endometrioid carcinoma. We retrospectively reviewed 185 cases of histologically confirmed endometrioid carcinoma by hysterectomy at Jichi Medical University Hospital between January 2006 and December 2011. We reviewed patient medical records to detect risk factors for lymph node metastasis to identify the optimum criteria for lymphadenectomy omission. Univariate analysis revealed risk factors for lymph node metastasis to be a large tumor size (volume index ≥40 cm³) (p<0.0001), tumor diameter >2 cm (p=0.0003), myometrial invasion ≥50% based on pre-operative MRI (p=0.0366), elevated serum CA125 (pre-menopausal value ≥70 U/mL, post-menopausal value ≥25 U/mL) (p=0.0004), and lymphadenopathy on pre-operative CT scans (p=0.0002). Multivariate analysis indicated that tumor volume index, tumor diameter, elevated serum CA125, and CT scans positive for lymphadenopathy were independent risk factors for lymph node metastasis. Thus, we set tumor diameter >2 cm, elevated serum CA125, and CT scans positive for lymphadenopathy as risk factors. In cases with no risk factors, the rate of lymph node metastasis was 2.1%, which rose to 8.9%, 30.4%, and 58.3% for those with one, two, and three risk factors, respectively. The rate of para-aortic lymph node metastasis rose from 0% to 2.5%, 10.9%, and 41.7% among those with zero, one, two, and three risk factors, respectively. We propose that lymphadenectomy can be omitted in cases of endometrioid carcinoma that do not have any of the following risk factors: tumor diameter >2 cm, elevated serum CA125, and a CT scan positive for lymphadenopathy. We believe that these new criteria will limit inter-institutional differences as they are all objective factors. Further, they are useful in predicting lymph node metastasis, including para-aortic lymph node metastasis, based on the number of risk factors present.


Estimating the Lambda measure in multiple-merger coalescents.
Multiple-merger coalescents, also known as Λ-coalescents, have been used to describe the genealogy of populations that have a skewed offspring distribution or that undergo strong selection. Inferring the characteristic measure Λ, which describes the rates of the multiple-merger events, is key to understand these processes. So far, most inference methods only work for some particular families of Λ-coalescents that are described by only one parameter, but not for more general models. This article is devoted to the construction of a non-parametric estimator of the density of Λ that is based on the observation at a single time of the so-called Site Frequency Spectrum (SFS), which describes the allelic frequencies in a present population sample. First, we produce estimates of the multiple-merger rates by solving a linear system, whose coefficients are obtained by appropriately subsampling the SFS. Then, we use a technique that aggregates the information extracted from the previous step through a kernel type of re-construction to give a non-parametric estimation of the measure Λ. We give a consistency result of this estimator under mild conditions on the behavior of Λ around 0. We also show some numerical examples of how our method performs.


Single-chain Fv/folate conjugates mediate efficient lysis of folate-receptor-positive tumor cells.
Bispecific antibodies that bind to a tumor antigen and the T cell receptor (TCR) redirect cytotoxic T lymphocytes (CTL) to lyse tumor cells which have escaped normal immune recognition mechanisms. One well-characterized tumor antigen, the folate receptor (FR), is expressed on most ovarian carcinomas and some types of brain cancer. Recently, it was shown that conjugates of folate and anti-TCR antibodies are extremely potent bispecific agents that target tumor cells expressing the high-affinity folate receptor, but not normal cells expressing only the reduced folate carrier protein. In this paper, it is shown that the size of these conjugates can be reduced to the smallest bispecific agent yet described (30 kDa) by attaching folate to a single-chain antibody, scFv, of the anti-TCR antibody KJ16. The scFv/folate conjugates are as effective as IgG/folate conjugates in mediating lysis of FR4 tumor cells by CTL. The optimal folate density was in the range of 5-15 folate molecules per scFv or IgG molecule, which yielded half-maximal lysis values (EC50) of approximately 40 pM (1.2 ng/mL for scFv). Finally, the scFv/folate conjugates could efficiently target tumor cells even in the presence of free folic acid at concentrations that are normally found in serum. Compared to conventional bispecific antibodies, the small size of scFv/folate conjugates may prove advantageous in the ability to penetrate tumors and in reduced immunogenicity.


A case of in-stent neoatherosclerosis 10 years after carotid artery stent implantation: observation with optical coherence tomography and plaque histological findings.
We report a patient's case of slow progressive in-stent restenosis 10 years after bare-metal stent implantation to his carotid artery. We treated the patient with an additional stent placement under a distal filter protection device. Optical coherence tomographic assessment and plaque histology during the carotid artery stenting (CAS) revealed atheromatous change at in-stent neointima, which contained lipid-rich plaque and calcification deposits. These findings suggest that in-stent neoatherosclerosis may play an important role in the pathogenesis of very late stent restenosis after CAS.


Patient and Public Involvement (PPI) in preclinical research: A scoping review protocol.
Introduction: Patient and public involvement (PPI) aims to improve the quality, relevance, and appropriateness of research and ensure that it meets the needs and expectations of those affected by particular conditions to the greatest possible degree. The evidence base for the positive impact of PPI on clinical research continues to grow, but the role of PPI in preclinical research (an umbrella term encompassing 'basic', 'fundamental', 'translational' or 'lab-based' research) remains limited. As funding bodies and policymakers continue to increase emphasis on the relevance of PPI to preclinical research, it is timely to map the PPI literature to support preclinical researchers involving the public, patients, or other service users in their research. Therefore, the aim of this scoping review is to explore the literature on patient and public involvement in preclinical research from any discipline. Methods: This scoping review will search the literature in Medline (PubMed), Embase, CINAHL, PsycINFO, Web of Science Core Collection, Scopus, and OpenGrey.net to explore the application of PPI in preclinical research. This review will follow the Joanna Briggs Institute (JBI) guidelines for scoping reviews. It will be reported according to the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR). Two reviewers will independently review articles for inclusion in the final review. Data extraction will be guided by the research questions. The PPI advisory panel will then collaboratively identify themes in the extracted data. Discussion: This scoping review will provide a map of current evidence surrounding preclinical PPI, and identify the body of literature on this topic, which has not been comprehensively reviewed to date. Findings will inform ongoing work of the research team, support the work of other preclinical researchers aiming to include PPI in their own research, and identify knowledge and practice gaps. Areas for future research will be identified.


Sugen-morphine model of pulmonary arterial hypertension.
Pulmonary arterial hypertension is a fatal disease associated with pulmonary vascular remodeling and right ventricular hypertrophy. Pre-clinical animal models that reproduce the human pulmonary arterial hypertension process and pharmacological response to available therapies are critical for future drug development. The most prevalent animal model reproducing many aspects of angioobliterative forms of pulmonary arterial hypertension is the rat Sugen/hypoxia model in which Sugen, a vascular endothelial growth factor receptor antagonist, primarily causes initiation of endothelial injury and later in the presence of hypoxia promotes proliferation of apoptosis-resistant endothelial cells. We previously demonstrated that exposure of human pulmonary microvascular endothelium to morphine and HIV-proteins results in initial apoptosis followed by increased proliferation. Here, we demonstrate that the double-hit of morphine and Sugen 5416 (Sugen-morphine) in rats leads to the development of pulmonary arterial hypertension with significant medial hypertrophy of pre-acinar pulmonary arteries along with neo-intimal thickening of intra-acinar vessels. In addition, the pulmonary smooth muscle and endothelial cells isolated from Sugen-morphine rats showed hyperproliferation and apoptotic resistance, respectively, in response to serum starvation. Our findings support that the dual hit model of Sugen 5416 and morphine provides another experimental strategy to induce significant pulmonary vascular remodeling and development of severe pulmonary arterial hypertension pathology in rats without exposure to hypoxia.


The programmed death-1 immune-suppressive pathway: barrier to antitumor immunity.
Programmed death ligand 1 (PD-L1, also known as B7 homolog 1 or CD274) is a major obstacle to antitumor immunity because it tolerizes/anergizes tumor-reactive T cells by binding to its receptor programmed death-1 (CD279), renders tumor cells resistant to CD8(+) T cell- and FasL-mediated lysis, and tolerizes T cells by reverse signaling through T cell-expressed CD80. PD-L1 is abundant in the tumor microenvironment, where it is expressed by many malignant cells, as well as by immune cells and vascular endothelial cells. The critical role of PD-L1 in obstructing antitumor immunity has been demonstrated in multiple animal models and in recent clinical trials. This article reviews the mechanisms by which PD-L1 impairs antitumor immunity and discusses established and experimental strategies for maintaining T cell activation in the presence of PD-L1-expressing cells in the tumor microenvironment.


Health service use costs by personality disorder following specialist and nonspecialist treatment: a comparative study.
The impact of specialist psychosocial treatment on health service use costs by patients with personality disorder is not yet sufficiently documented. In this prospective study we compare patterns of health service costs by three groups of people with personality disorder treated in a hospital-based program (IPP), a step down program (SDP), and a general psychiatric program (GPP). Total service use costs at follow up, compared with intake costs, showed that significantly higher savings were achieved by SDP and IIP compared with GPP. Cost reductions in SDP were significantly greater than in IPP. Significant cost reductions were found between treatment programs in social worker and community psychiatric nursing and psychotherapy. The cost-effectiveness of the two specialist treatment programs was indicated by the significant association between total cost reduction and clinical outcome in GPP and IPP, but not in GPP. The effect of Major Depression and Borderline Personality Disorder on health service use alone and in combination was also investigated. The finding that Major Depression was found to be more significantly associated with higher health service use costs than Borderline Personality Disorder is discussed.


Effect of growth in serum on uptake of Neisseria gonorrhoeae by human neutrophils.
Because serum exposure precedes interaction of invasive Neisseria gonorrhoeae with neutrophils, neutrophil association with gonococci grown in serum was assessed. Uptake of a nonpiliated serum-resistant strain grown in 20% serum was reduced to 63.5% of control values. Heated serum (20%) yielded similar results. Stimulation of hexose monophosphate shunt activity in neutrophils by heated serum-grown gonococci (a phenomenon reflecting phagocytosis) was 64.5% of control values. Because gonococcal outer membrane (OM) structures mediate interaction with neutrophils, lithium acetate-treated OM preparations of gonococci grown in heated serum were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Multiple new protein bands and a quantitative decrease in amounts of proteins I, II, and III were noted. However, a decrease in the amounts of these membrane proteins was not observed with alternative membrane extraction techniques. Gonococcal growth in 20% serum dialyzed in 3,500-molecular-weight exclusion tubing allowed normal neutrophil association and OM protein expression.


Post-translational myristoylation at the cross roads of cell death, autophagy and neurodegeneration.
In a little more than a decade, post-translational myristoylation (PTMyr) has become an established post-translational modification during cell death. It involves the addition of the fatty acid myristate to newly exposed N-terminal glycines following caspase cleavage. It promotes membrane binding and relocalization of functional protein domains released by caspase cleavage during apoptosis, or programmed cell death. However, as the requirement of caspase cleavage has expanded beyond just cell death, it has become apparent that PTMyr may play a role in cell survival, differentiation and now autophagy. Herein, we describe how myristoylation may play a role in autophagy with an emphasis on PTMyr.


Rapid treatment of mild acne with a novel skin care system containing 1% salicylic acid, 10% buffered glycolic acid, and botanical ingredients.
The biggest hurdle in the treatment of acne vulgaris is patient non-compliance that is due in large part to poor tolerability to common acne medications. As such, new acne treatments must be developed that balance good anti-acne efficacy with excellent tolerability in order to ensure patient adherence and by extension ensure good clinical outcomes. The goal of the present study was to determine the tolerability and efficacy of a novel skin care system, composed of a cleanser, containing 1% salicylic acid and botanical ingredients, and a treatment gel, containing 1% salicylic acid, 10% buffered glycolic acid and botanical ingredients for the treatment of mild acne. In this single-center, open-label clinical study, 25 male and female volunteers used the test cleanser and test gel twice daily over six weeks. Tolerability assessments showed that the skin care regimen was very well tolerated by all study volunteers. Acne severity was significantly reduced by two acne grades at six weeks. Inflammatory lesion counts were significantly reduced, on average, by 59.06% (P ≤ 0.0001), 91.62% (P ≤ 0.0001), 90.85% (P ≤ 0.0001) and by 98.55% (P ≤ 0.0001) at weeks 1, 2, 4, and 6, respectively. Non-inflammatory lesion counts were reduced, on average, by 13.54% (ns), 38.95% (P ≤ 0.0001), 44.48% (P ≤ 0.0001), and by 56.10% (P ≤ 0.0001) at weeks 1, 2, 4, and 6, respectively. Standardized photography also demonstrated a progressive reduction in acne lesions over time. In conclusion, results of the present study suggest that the tested skin care regimen offers rapid acne clearance and excellent tolerability that together may help to improve patient adherence as well as treatment outcome.


The Arabidopsis BAP1 and BAP2 genes are general inhibitors of programmed cell death.
Here we identify the BAP1 and BAP2 genes of Arabidopsis (Arabidopsis thaliana) as general inhibitors of programmed cell death (PCD) across the kingdoms. These two homologous genes encode small proteins containing a calcium-dependent phospholipid-binding C2 domain. BAP1 and its functional partner BON1 have been shown to negatively regulate defense responses and a disease resistance gene SNC1. Genetic studies here reveal an overlapping function of the BAP1 and BAP2 genes in cell death control. The loss of BAP2 function induces accelerated hypersensitive responses but does not compromise plant growth or confer enhanced resistance to virulent bacterial or oomycete pathogens. The loss of both BAP1 and BAP2 confers seedling lethality mediated by PAD4 and EDS1, two regulators of cell death and defense responses. Overexpression of BAP1 or BAP2 with their partner BON1 inhibits PCD induced by pathogens, the proapoptotic gene BAX, and superoxide-generating paraquat in Arabidopsis or Nicotiana benthamiana. Moreover, expressing BAP1 or BAP2 in yeast (Saccharomyces cerevisiae) alleviates cell death induced by hydrogen peroxide. Thus, the BAP genes function as general negative regulators of PCD induced by biotic and abiotic stimuli including reactive oxygen species. The dual roles of BAP and BON genes in repressing defense responses mediated by disease resistance genes and in inhibiting general PCD has implications in understanding the evolution of plant innate immunity.


COVID-19 Mortality At The Neighborhood Level: Racial And Ethnic Inequalities Deepened In Minnesota In 2020.
Substantial racial and ethnic disparities in COVID-19 mortality have been observed at the state and national levels. However, less is known about how race and ethnicity and neighborhood-level disadvantage may intersect to contribute to both COVID-19 mortality and excess mortality during the pandemic. To assess this potential interaction of race and ethnicity with neighborhood disadvantage, we link death certificate data from Minnesota from the period 2017-20 to the Area Deprivation Index to examine hyperlocal disparities in mortality. Black, Indigenous, and people of color (BIPOC) standardized COVID-19 mortality was 459 deaths per 100,000 population in the most disadvantaged neighborhoods compared with 126 per 100,000 in the most advantaged. Total mortality increased in 2020 by 14 percent for non-Hispanic White people and 41 percent for BIPOC. Statistical decompositions show that most of this growth in racial and ethnic disparity is associated with mortality gaps between White people and communities of color within the same levels of area disadvantage, rather than with the fact that White people live in more advantaged areas. Policy interventions to reduce COVID-19 mortality must consider neighborhood context.


Inhibitory effects of fatty acids on the activity of mushroom tyrosinase.
The effects of fatty acids, octanoic acid, (2E, 4E)-hexa-2,4-dienoic acid, hexanoic acid, (2E)-but-2-enoic acid, and butyric acid on the activities of mushroom tyrosinase have been investigated. The results showed that the fatty acids can potently inhibit both monophenolase activity and diphenolase activity of tyrosinase, and that the unsaturated fatty acids exhibited stronger inhibitory effect against tyrosinase than the corresponding saturated fatty acids, and the inhibitory effects were enhanced with the extendability of the fatty acid chain. For the monophenolase activity, the fatty acids could not only lengthen the lag period, but also decrease the steady-state activities. For the diphenolase activity, fatty acids displayed reversible inhibition. Kinetic analyses showed that octanoic acid and hexanoic acid were mixed-type inhibitors and (2E,4E)-hexa-2,4-dienoic acid and (2E)-but-2-enoic acid were noncompetitive inhibitors. The inhibition constants have been determined and compared.


A Novel Conservative Approach Combining "SealBio" and "Surgical Fenestration" for Healing of Large Periapical Cystic Lesions of Endodontic Origin: A Pilot Study.
To evaluate the treatment outcome of large periapical cystic lesions treated by combining two novel, conservative approaches, "SealBio" and "Surgical Fenestration". Five cases (4M:1F, age range 14-38 years, mean age 24.5 years) of large periapical cystic lesions, diagnosed on clinical and radiographic examination, were included in the study. After informed consent, endodontic treatment was initiated; chemo-mechanical preparation and intra-canal dressing of calcium hydroxide was given. At the next sitting after one week, further disinfection root canals was done by "apical clearing", "apical foramen widening" and irrigation. A cotton pellet was kept in the access cavity. After local anaesthesia, full thickness muco-periosteal flap was reflected and the thinned out bone was removed with bone rongers, a small piece of cystic lining was excised and the cystic cavity was copiously flushed with Betadine solution. The remaining cystic lining was gently curetted and the flap was sutured back. "SealBio" was performed after gentle irrigation with saline and intentional over instrumentation. A calcium sulphate based cement was pushed in the cervical third of the canal and the access opening was sealed with glass ionomer cement. Patient was prescribed antibiotics and anti-inflammatory drugs for 5 days and sutures were removed after 7 days. Patients were followed up clinically and radiographically at regular intervals. In this pilot study, treatment outcome after combined technique of "SealBio" and "Surgical fenestration" was found to be highly effective in healing of large periapical cystic lesions. It was simple to perform and very conservative treatment; it required minimal bone removal, obviated the need for complete cyst enucleation, apicectomy and retrograde filling.


Temperament in young adulthood and later mortality: prospective observational study.
To determine the association between a clinician assessment of temperament in early adulthood and cause specific mortality. Prospective observational study. Glasgow University. 9239 male former students aged 16-30 (mean 20.5) years who participated in an ongoing health survey from 1948-68. A physician recorded free text assessment of temperament, which seemed to capture aspects of personality (trait) and mental health (state), was coded into: stable, anxious, schizoid, hypomanic, odd, depressed, immature, hypochondriacal, unstable, and obsessive. Associations between temperament and mortality were investigated using Cox proportional hazards models. There were 878 deaths. Most students-8342 (90.3%)-were assessed as stable, the remaining 897 (9.7%) having at least one, and 103 (1.1%) having more than one, temperament type. The second most common temperament was anxiety, recorded in 520 (5.6%) students. In multivariable analyses, having at least one temperament type was associated with increased all cause and stroke mortality, hazard ratios (95% confidence intervals): 1.23 (1.01 to 1.50) and 1.95 (1.06 to 3.59) respectively, compared with stable students. Students with more than one temperament type had higher risk of death from: all causes, 2.05 (1.36 to 3.09); stroke, 3.26 (1.01 to 10.56); and cancer, 2.90 (1.62 to 5.20). Anxiety was positively associated with all cause and cancer mortality, respective hazard ratios: 1.36 (1.07 to 1.72) and 1.51 (1.04 to 2.20). Men labelled hypomanic had increased cardiovascular mortality risk, 1.90 (1.05 to 3.44). Markers of early adult psychological distress are associated with increased mortality. Mechanisms underlying these associations require investigation.


Combined effects of lead and acid rain on photosynthesis in soybean seedlings.
To explore how lead (Pb) and acid rain simultaneously affect plants, the combined effects of Pb and acid rain on the chlorophyll content, chlorophyll fluorescence reaction, Hill reaction rate, and Mg(2+)-ATPase activity in soybean seedlings were investigated. The results indicated that, when soybean seedlings were treated with Pb or acid rain alone, the chlorophyll content, Hill reaction rate, Mg(2+)-ATPase activity, and maximal photochemical efficiency (F(v)/F(m)) were decreased, while the initial fluorescence (F 0) and maximum quantum yield (Y) were increased, compared with those of the control. The combined treatment with Pb and acid rain decreased the chlorophyll content, Hill reaction rate, Mg(2+)-ATPase activity, F(v)/F(m), and Y and increased F 0 in soybean seedlings. Under the combined treatment with Pb and acid rain, the two factors showed additive effects on the chlorophyll content in soybean seedlings and exhibited antagonistic effects on the Hill reaction rate. Under the combined treatment with high-concentration Pb and acid rain, the two factors exhibited synergistic effects on the Mg(2+)-ATPase activity, F 0, F v/F m, as well as Y. In summary, the inhibition of the photosynthetic process is an important physiological basis for the simultaneous actions of Pb and acid rain in soybean seedlings.


A new method of extrapolating the sweep pattern visual evoked potential acuity.
In order to compare the amplitude-spatial frequency (A-SP) regression method with amplitude-logVA (A-logVA) regression methods in extrapolating the sweep pattern visual evoked potential (SPVEP) acuity. We measured SPVEPs in 21 children and three adults using sinusoidally-modulated horizontal gratings as stimuli. The responses were averaged and displayed through discrete Fourier transformations. SPVER acuity was then estimated by using both the SPVEP amplitude- spatial frequency function (A-SP function regression method) and the SPVEP amplitude-log visual-angle function (A-logVA function regression method). Furthermore, the Bailey Lovie logMAR chart was employed to define visual acuity. Curve estimates were calculated to derive a correlation index (R) for each method. There are significant differences (t = 2.71, P < 0.05) between the correlation indices of curves obtained using the A-logVA function (logarithmic model, 0.95 +/- 0.01) and that obtained by the A-SP function (inverse model, 0.92 +/- 0.02). The overall correlation coefficient (r) between logMAR acuity and acuity calculated by the A-logVA regression method was 0.32 (P < 0.05). The overall correlation coefficient (r) between logMAR acuity and acuity calculated by the A-SP regression method was 0.41 (P < 0.05). Paired t-tests show that SPVEP acuity from the A-logVA function was not significantly different from acuities of the logMAR function (t = 1.77, P = 0.09). The difference in their mean values is 0.14 +/- 0.08. However, SPVEP acuity calculated using the A-SP function regression method is significantly different from the acuity calculated from the logMAR function (t = 10.09, P < 0.01). The difference in their mean values is 0.41 +/- 0.04. The amplitude-logVA function regression method is more accurate in estimating SPVEP acuity in normal subjects with good visual acuity.


PP2A inhibition causes synthetic lethality in BRCA2-mutated prostate cancer models via reactivating spindle assembly checkpoint.
Mutations in the BRCA2 tumor suppressor gene have been associated with an increased risk of developing prostate cancer. One of the paradoxes concerning BRCA2 is the fact that its inactivation affects genetic stability and is deleterious for cellular and organismal survival, while BRCA2-mutated cancer cells adapt to this detriment and malignantly proliferate. Therapeutic strategies for tumors arising from BRCA2 mutations may be discovered by understanding these adaptive mechanisms. In this study, we conducted forward genetic synthetic viability screenings in C. elegans brc-2 (Cebrc-2) mutants and found that Ceubxn-2 inactivation rescued the viability of Cebrc-2 mutants. Moreover, loss of NSFL1C, the mammalian ortholog of CeUBXN-2, suppressed the spindle assembly checkpoint (SAC) activation and promoted the survival of BRCA2-deficient cells. Mechanistically, NSFL1C recruited USP9X to inhibit the polyubiquitination of AURKB and reduce the removal of AURKB from the centromeres by VCP, which is essential for SAC activation. SAC inactivation is common in BRCA2-deficient prostate cancer patients, but PP2A inhibitors could reactivate the SAC and achieve BRCA2-deficient prostate tumor synthetic lethality. Our research reveals the survival adaptation mechanism of BRCA2-deficient prostate tumor cells and provides different angles for exploring synthetic lethal inhibitors in addition to targeting DNA damage repair pathways.


Antisense oligodeoxynucleotides for urokinase-plasminogen activator receptor have anti-invasive and anti-proliferative effects in vitro and inhibit spontaneous metastases of human melanoma in mice.
We have targeted the urokinase-type plasminogen activator receptor (uPAR) with phosphorothioate antisense oligonucleotides (aODN) in vitro to evaluate the anti-invasive and anti-proliferative effects of uPAR down-regulation, as well as in vivo to evaluate anti-tumor and anti-metastatic activity. aODN-dependent uPAR downregulation in vitro was induced in cells of human melanoma, mammary carcinoma, ovarian carcinoma and SV-40-transformed embryonic lung fibroblasts. uPAR was determined by an antibody-based assay and by semiquantitative polymerase chain reaction. Cell invasion was evaluated by Matrigel invasion assay and cell proliferation by direct cell counting. aODN reduced uPAR, invasion and proliferation in all the treated cell lines. Following aODN treatment, human melanoma cells exhibited a strong decrease of uPAR-dependent ERK1/2 activation and were used in vivo to control metastasis in CD-1 male nude (nu/nu) mice by uPAR aODN injection. 60 mice were injected in the hind leg muscles with a suspension of 10(6) melanoma cells. After 4 days, when a tumor mass of about 350 mg was evident in all the mice injected, 20 mice were treated i.v. with aODN and 20 with dODN at 0.5 mg/day for 5 consecutive days. Twenty control mice were not treated. A second and third cycle of treatment was administered at 2-day intervals. Treatment with aODN resulted into a 78% reduction of lung metastases and 45% reduction of the primary tumor mass with no loss of body weight. Our results suggest to evaluate the utility of uPAR aODN in controlling the metastatic spreading of human melanoma.


Microbial source tracking and spatial analysis of E. coli contaminated private well waters in southeastern Ontario.
Private water supplies, which are the primary source of drinking water for rural communities in developed countries, are at risk of becoming fecally contaminated. It is important to identify the source of contamination in order to better understand and address this human health risk. Microbial source tracking methods using human, bovine and general Bacteroidales markers were performed on 716 well water samples from southeastern Ontario, which had previously tested positive for Escherichia coli. The results were then geospatially analyzed in order to elucidate contamination patterns. Markers for human feces were found in nearly half (49%) of all samples tested, and a statistically significant spatial cluster was observed. A quarter of the samples tested positive for only general Bacteroidales markers (25.7%) and relatively few bovine specific marker positives (12.6%) were found. These findings are fundamental to the understanding of pathogen dynamics and risk in the context of drinking well water and will inform future research regarding host-specific pathogens in private well water samples.


Disrupted-in-Schizophrenia-1 (Disc1) is necessary for migration of the pyramidal neurons during mouse hippocampal development.
The hippocampus has a highly ordered structure and is composed of distinct layers. Neuronal migration is an essential part of the process of the layer formation because neurons are primarily generated near the ventricle and must migrate to arrive at their final locations during brain development. Impairment of brain development is thought to underlie the etiology of psychiatric disorders. Consistent with this idea, many genetic risk factors for psychiatric disorders play critical roles during brain development. As one example, Disrupted-in-Schizophrenia-1 (DISC1) is a genetic risk factor for major psychiatric disorders and plays various roles during neurodevelopment. To examine the role of Disc1 in the hippocampal development, we suppressed expression of Disc1 in the CA1 region of the developing mouse hippocampus by using the RNA interference (RNAi) technology and an in utero electroporation system. Disc1 suppression was found to impair migration of the CA1 pyramidal neurons. This effect was especially apparent while the majority of the transfected neurons were passing through the stratum pyramidale of the developing hippocampus. The migration of neurons was restored by expression of an RNAi-resistant wild-type mouse Disc1, indicating that the migration defect was caused by specific suppression of Disc1. In the mature hippocampus, the migration defect resulted in malposition and disarray of the pyramidal neurons. These findings indicate that Disc1 is required for migration and layer formation by the CA1 pyramidal neurons during hippocampal development.


Temporal broadening of optical pulses propagating through non-Kolmogorov turbulence.
General formulations of the temporal averaged pulse intensity for optical pulses propagating through either non-Kolmogorov or Kolmogorov turbulence are deduced under the strong fluctuation conditions and the narrow-band assumption. Based on these formulations, an analytical formula for the turbulence-induced temporal half-width of spherical-wave Gaussian (SWG) pulses is derived, and the single-point, two-frequency mutual coherence function (MCF) of collimated Gaussian-beam waves in atmospheric turbulence is formulated analytically, by which the temporal averaged pulse intensity of collimated space-time Gaussian (CSTG) pulses can be calculated numerically. Calculation results show that the temporal broadening of both SWG and CSTG pulses in atmospheric turbulence depends heavily on the general spectral index of the spatial power spectrum of refractive-index fluctuations, and the temporal broadening of SWG pulses can be used to approximate that of CSTG pulses on the axis with the same turbulence parameters and propagation distances. It is also illustrated by numerical calculations that the variation in the turbulence-induced temporal half-width of CSTG pulses with the radial distance is really tiny.