Chondrocytes dedifferentiated by serial monolayer culture form cartilage nodules in nude mice.
Chondrocytes isolated from rabbit costal growth cartilage grew rapidly and expressed the differentiated phenotypes of chondrocytes in primary monolayer cultures. They had a typical polygonal shape, synthesized glycosaminoglycan (GAG) actively and responded to parathyroid hormone (PTH). However, during successive passages they lost the abilities to proliferate and express differentiated phenotypes. They changed from polygonal to fibroblast-like morphology during the third to fourth passage. Their GAG synthesis rapidly decreased in the 4th passage to 10% of that during primary culture. The increase in GAG synthesis and intracellular cyclic AMP induced by PTH also became smaller and were lost in the 4th passage. From these findings, cells in the 4th passage appeared to be dedifferentiated. However, subcutaneous transplantation of cells at the 5th passage into nude mice resulted in the formation of cartilaginous nodules. Moreover, cells isolated from these nodules and cultured in vitro had a typical polygonal shape and synthesized GAG actively and PTH increased their metachromatic matrix and intracellular cyclic AMP level. These findings suggest that chondrocytes that appear to be dedifferentiated after serial passages still have the potential to differentiate.


Strain-Induced Spin Crossover in Phthalocyanine-Based Organometallic Sheets.
Motivated by the recent success in synthesizing two-dimensional Fe-phthalocyanine (poly-FePc) porous sheets, we studied strain-induced spin crossover in poly-TMPc (TM = Mn, Fe, Co, and Ni) systems by using first-principle calculations based on density functional theory. A small amount of biaxial tensile strain is found to not only significantly enhance the magnetic moment of the central TM atoms by 2 μB when the strain reaches a critical value, but also the systems undergo low-spin (LS) to high-spin (HS) transition. These systems, however, show different response to strain, namely, poly-FePc sheet becomes ferromagnetic (FM) while poly-MnPc and poly-NiPc sheets become antiferromagnetic (AFM). Poly-CoPc, on the other hand, remains AFM. These predicted results can be observed in suspended poly-TMPc sheets by using scanning tunneling microscope (STM) tips to manipulate strain.


Effects of Enzyme Complex Supplementation to a Paddy-based Diet on Performance and Nutrient Digestibility of Meat-type Ducks.
Paddy rice is rarely used as a feed because of its high fiber content. In this study, two experiments were conducted to study the effects of supplementing an enzyme complex consisting of xylanase, beta-glucanase and cellulase, to paddy-based diets on the performance and nutrient digestibility in meat-type ducks. In the both experiments, meat-type ducks (Cherry Valley) were randomly assigned to four treatments. Treatment 1 was a basal diet of corn-soybean; treatment 2 was a basal diet of corn-paddy-soybean; treatment 3, had enzyme complex added to the corn-paddy-soybean basal diet at levels of 0.5 g/kg diet; and treatment 4, had enzyme complex added to the corn-paddy-soybean diet at levels of 1.0 g/kg diet. The results showed that the enzyme complex increased the ADG, and decreased the ADFI and F/G significantly (p<0.05) in the ducks, and the ADFI for the ducks fed the corn-paddy-soybean diet showed no difference compared to the ducks fed corn-soybean diets at all stages of the experiment (p<0.05). When corn was partially replaced by paddy, the digestibility of CP and NDF was decreased and increased, respectively (p<0.05), and the level of enzyme complex had a significant effect on both CP and NDF digestibility (p<0.05). As for the AME, addition of enzyme complex increased it significantly (p<0.05), but both diet types and levels of enzyme complex had no effect (p>0.05). The outcome of this research indicates that the application of enzyme complex made up of xylanase, beta-glucanase, and cellulase, in the corn-paddy-soybean diet, can improve performance and nutrition digestibility in meat-type ducks.


Analysis of a meiosis-specific URS1 site: sequence requirements and involvement of replication protein A.
URS1 is a transcriptional repressor site found in the promoters of a wide variety of yeast genes that are induced under stress conditions. In the context of meiotic promoters, URS1 sites act as repressor sequences during mitosis and function as activator sites during meiosis. We have investigated the sequence requirements of the URS1 site of the meiosis-specific HOP1 gene (URS1H) and have found differences compared with a URS1 site from a nonmeiotic gene. We have also observed that the sequence specificity for meiotic activation at this site differs from that for mitotic repression. Base pairs flanking the conserved core sequence enhance meiotic induction but are not required for mitotic repression of HOP1. Electrophoretic mobility shift assays of mitotic and meiotic cell extracts show a complex pattern of DNA-protein complexes, suggesting that several different protein factors bind specifically to the site. We have determined that one of the complexes of URS1H is formed by replication protein A (RPA). Although RPA binds to the double-stranded URS1H site in vitro, it has much higher affinity for single-stranded than for double-stranded URS1H, and one-hybrid assays suggest that RPA does not bind to this site at detectable levels in vivo. In addition, conditional-lethal mutations in RPA were found to have no effect on URS1H-mediated repression. These results suggest that although RPA binds to URS1H in vitro, it does not appear to have a functional role in transcriptional repression through this site in vivo.


Influence of creatine supplementation on bone quality in the ovariectomized rat model: an FT-Raman spectroscopy study.
The influence of creatine (Cr) supplementation on cortical and trabecular bone from ovariectomized rats was studied using FT-Raman spectroscopy. The intensity of organic-phase Raman bands was compared to mineral phase ones. Twenty-one female Wistar rats aged 3 months were divided into three groups (n = 7 per group): ovariectomized (OVX), ovariectomized treated with creatine (CRE) and sham-operated (SHAM) groups. Creatine supplementation (300 mg kg(-1) day(-1)) was provided for 8 weeks, starting 12 weeks after ovariectomy. FT-Raman spectroscopy was performed on the right medial femoral mid-shaft (cortical bone) and third lumbar vertebral body (trabecular bone). The integrated intensities of mineral phase (phosphate and carbonate bands at 959 and 1,071 cm(-1), respectively) and organic phase (amide I band at 1,665 cm(-1)) Raman bands were analyzed. The mineral-to-matrix (phosphate/amide I), carbonate-to-phosphate, and carbonate-to-amide I ratios were analyzed to assess bone quality. The phosphate content on trabecular bone was higher in the CRE group than the OVX group (p < 0.05). No significant changes in mineral or organic phases on cortical bone were observed. A radiographic assessment of bone density was encouraging as the same findings were showed by Raman intensity of phosphate from cortical (r(2) = 0.8037) and trabecular bones (r(2) = 0.915). Severe ovariectomy-induced bone loss was confirmed by FT-Raman spectroscopy. The results suggest that the chemical composition of trabecular bone tissue may be positively influenced by Cr supplementation after ovariectomy.


High-throughput assays using a luciferase-expressing replicon, virus-like particles, and full-length virus for West Nile virus drug discovery.
Many flaviviruses cause significant human disease worldwide. The development of flavivirus chemotherapy requires reliable high-throughput screening (HTS) assays. Although genetic systems have been developed for many flaviviruses, their usage in antiviral HTS assays has not been well explored. Here we compare three cell-based HTS assays for West Nile virus (WNV) drug discovery: (i) an assay that uses a cell line harboring a persistently replicating subgenomic replicon (containing a deletion of viral structural genes), (ii) an assay that uses packaged virus-like particles containing replicon RNA, and (iii) an assay that uses a full-length reporting virus. A Renilla luciferase gene was engineered into the replicon or into the full-length viral genome to monitor viral replication. Potential inhibitors could be identified through suppression of luciferase signals upon compound incubation. The antiviral assays were optimized in a 96-well format, validated with known WNV inhibitors, and proved useful in identifying a new inhibitor(s) through HTS of a compound library. In addition, because each assay encompasses multiple but discrete steps of the viral life cycle, the three systems could potentially be used to discriminate the mode of action of any inhibitor among viral entry (detected by assays ii and iii but not by assay i), replication (including viral translation and RNA synthesis; detected by assays i to iii), and virion assembly (detected by assay iii but not by assays i and ii). The approaches described in this study should be applicable to the development of cell-based assays for other flaviviruses.


Bone marrow stem cells and their niche components are adversely affected in advanced cirrhosis of the liver.
Bone marrow (BM) is a reservoir for immune and hematopoietic cells and critical for tissue repair and regeneration. All of these functions are severely altered in cirrhosis. We investigated the cellular and functional state of BM in cirrhosis patients. We studied the histological, cellular, and molecular changes in BM of cirrhosis patients (n = 168) and controls (n = 44). Hematopoietic stem cells (HSCs) and associated niche cells, mesenchymal stem cells, Schwann cells, neural fibers, and endothelial cells were evaluated by immunohistochemistry. Cytokines and growth factors were analyzed in peripheral blood and BM plasma. Cirrhotic BM showed an inverse correlation between cluster of differentiation 34+HSCs and Model of End-Stage Liver Disease (ρ = -0.582, P < 0.001) and Child's scores (P < 0.038). BMs of cirrhosis patients with higher Model of End-Stage Liver Disease (>15) showed significantly decreased HSCs, mesenchymal stem cells, Schwann cells, and neural fibers; increased interleukin-1β (P = 0.004), tumor necrosis factor-α (P = 0.040), and interferon-γ (P = 0.03); and decreased oncostatin M (P = 0.04), stem cell factor (P = 0.05), and stromal cell-derived factor 1 (P = 0.03) compared to those with lower Model of End-Stage Liver Disease scores (≤15). The cluster of differentiation 34+ cell population was a predictor for the development of sepsis (P < 0.001), and per unit loss increased the probability of sepsis by 16%. Cirrhosis patients with fewer HSCs had lower hemoglobin (P = 0.05) and platelet counts (P = 0.05) and showed early graft dysfunction. Increasing severity of cirrhosis causes derangement of the hematopoietic niche and loss of HSCs, contributing to the hematological and immunological dysfunctions and reduced potential for regeneration; restoring BM functions could provide new therapeutic options in cirrhosis. (Hepatology 2016;64:1273-1288).


In vitro synergism of magnolol and honokiol in combination with antibacterial agents against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA).
Methicillin-resistant Staphylococcus aureus (MRSA) is a problematic pathogen posing a serious therapeutic challenge in the clinic. It is often multidrug-resistant (MDR) to conventional classes of antibacterial agents and there is an urgent need to develop new agents or strategies for treatment. Magnolol (ML) and honokiol (HL) are two naturally occurring diallylbiphenols which have been reported to show inhibition of MRSA. In this study their synergistic effects with antibacterial agents were further evaluated via checkerboard and time-kill assays. The susceptibility spectrum of clinical MRSA strains was tested by the disk diffusion method. The minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of ML and HL were assayed by broth microdilution. The synergy was evaluated through checkerboard microdilution and time-killing experiments. ML and HL showed similar activity against both MSSA and MRSA with MIC/MBC at 16 ~ 64 mg/L, with potency similar to amikacin (AMK) and gentamicin (GEN). When they were used in combination with conventional antibacterial agents, they showed bacteriostatic synergy with FICIs between 0.25 ~ 0.5, leading to the combined MICs decreasing to as low as 1 ~ 2 and 1 ~ 16 mg/L for ML (HL) and the agents, respectively. MIC50 of the combinations decreased from 16 mg/L to 1 ~ 4 mg/L for ML (HL) and 8 ~ 128 mg/L to 2 ~ 64 mg/L for the antibacterial agents, which exhibited a broad spectrum of synergistic action with aminoglycosides (AMK, etilmicin (ETM) and GEN), floroquinolones (levofloxacin (LEV), ciprofloxacin and norfloxacin), fosfomycin (FOS) and piperacillin. The times of dilution (TOD, the extent of decreasing in MIC value) were determined up to 16 for the combined MIC. A more significant synergy after combining was determined as ML (HL) with AMK, ETM, GEN and FOS. ML (HL) combined with antibacterial agents did not show antagonistic effects on any of the ten MRSA strains. Reversal effects of MRSA resistance to AMK and GEN by ML and HL were also observed, respectively. All the combinations also showed better dynamic bactericidal activity against MRSA than any of single ML (HL) or the agents at 24 h incubation. The more significant synergy of combinations were determined as HL (ML) + ETM, HL + LEV and HL + AMK (GEN or FOS), with △LC24 of 2.02 ~ 2.25. ML and HL showed synergistic potentiation of antibacterial agents against clinical isolates of MRSA and warrant further pharmacological investigation.


SARS-CoV-2 spike protein S1 induces fibrin(ogen) resistant to fibrinolysis: implications for microclot formation in COVID-19.
Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2)-induced infection, the cause of coronavirus disease 2019 (COVID-19), is characterized by unprecedented clinical pathologies. One of the most important pathologies, is hypercoagulation and microclots in the lungs of patients. Here we study the effect of isolated SARS-CoV-2 spike protein S1 subunit as potential inflammagen sui generis. Using scanning electron and fluorescence microscopy as well as mass spectrometry, we investigate the potential of this inflammagen to interact with platelets and fibrin(ogen) directly to cause blood hypercoagulation. Using platelet-poor plasma (PPP), we show that spike protein may interfere with blood flow. Mass spectrometry also showed that when spike protein S1 is added to healthy PPP, it results in structural changes to β and γ fibrin(ogen), complement 3, and prothrombin. These proteins were substantially resistant to trypsinization, in the presence of spike protein S1. Here we suggest that, in part, the presence of spike protein in circulation may contribute to the hypercoagulation in COVID-19 positive patients and may cause substantial impairment of fibrinolysis. Such lytic impairment may result in the persistent large microclots we have noted here and previously in plasma samples of COVID-19 patients. This observation may have important clinical relevance in the treatment of hypercoagulability in COVID-19 patients.


Cytotoxicity of human recombinant arginase I (Co)-PEG5000 in the presence of supplemental L-citrulline is dependent on decreased argininosuccinate synthetase expression in human cells.
Human recombinant arginase I cobalt [HuArgI (Co)] coupled with polyethylene glycol 5000 [HuArgI (Co)-PEG5000] has shown potent in-vitro depletion of arginine from tissue culture medium. We now show that HuArgI (Co)-PEG5000 is toxic to almost all cancer cell lines and to some normal primary cells examined. In contrast, HuArgI (Co)-PEG5000 in combination with supplemental L-citrulline is selectively cytotoxic to a fraction of human cancer cell lines in tissue culture, including some melanomas, mesotheliomas, acute myeloid leukemias, hepatocellular carcinomas, pancreas adenocarcinomas, prostate adenocarcinomas, lung adenocarcinomas, osteosarcomas, and small cell lung carcinomas. Unfortunately, a subset of normal human tissues is also sensitive to HuArgI (Co)-PEG5000 with L-citrulline supplementation, including umbilical endothelial cells, bronchial epithelium, neurons, and renal epithelial cells. We further show that cell sensitivity is predicted by the level of cellular argininosuccinate synthetase protein expression measured by immunoblots. By comparing a 3-day and 7-day exposure to HuArgI (Co)-PEG5000 with supplemental L-citrulline, some tumor cells sensitive on short-term assay are resistant in the 7-day assay consistent with the induction of argininosuccinate synthetase expression. On the basis of these results, we hypothesize that HuArgI (Co)-PEG5000 in combination with L-citrulline supplementation may be an attractive therapeutic agent for some argininosuccinate synthetase-deficient tumors. These in-vitro findings stimulate further development of this molecule and may aid in the identification of tissue toxicities and better selection of patients who will potentially respond to this combination therapy.


Decreased binding to proteins and cells of polymeric gene delivery vectors surface modified with a multivalent hydrophilic polymer and retargeting through attachment of transferrin.
Binding of serum proteins to polyelectrolyte gene delivery complexes is thought to be an important factor limiting bloodstream circulation and restricting access to target tissues. Protein binding can also inhibit transfection activity in vitro. In this study a multivalent reactive hydrophilic polymer has been used to inhibit protein binding. This polymer is based on poly-[N-(2-hydroxypropyl)methacrylamide] (pHPMA) bearing pendent oligopeptide (Gly-Phe-Leu-Gly) side chains terminated in reactive 4-nitrophenoxy groups (8.6 mol%). The polymer reacts with the primary amino groups of poly(L-lysine) (pLL) and produces a hydrophilic coating on the surface of pLL.DNA complexes (as measured by fluorescamine). The resulting pHPMA-coated complexes show a decreased surface charge (from +14 mV for pLL.DNA complexes to -25 mV for pHPMA-modified complexes) as measured by zeta potential analysis. The pHPMA-coated complexes also show a slightly increased average diameter (approximately 90 nm compared with 60 nm for pLL. DNA complexes) as viewed by atomic force and transmission electron microscopy and around 100 nm as viewed by photon correlation spectroscopy. They are completely resistant to protein interaction, as determined by turbidometry and SDS-polyacrylamide gel electrophoresis analysis of complexes isolated from plasma, and show significantly decreased nonspecific uptake into cells in vitro. Spare reactive ester groups can be used to conjugate targeting ligands (e.g. transferrin) on to the surface of the complex to provide a means of tissue-specific targeting and transfection. The properties of these complexes therefore make them promising candidates for targeted gene delivery, both in vitro and potentially in vivo.


Stress, social behaviour, and secondary sexual traits in a male primate.
We examined variation in glucocorticoid levels in the mandrill, a brightly coloured primate species, to identify major social influences on stress hormones, and investigate relationships among glucocorticoid levels, testosterone and secondary sexual ornamentation. We collected a total of 317 fecal samples for 16 adult male mandrills over 13 months, including mating and non-mating periods and periods of both dominance rank stability and instability, and compared fecal glucocorticoid levels with dominance rank, rank stability, presence of receptive females, gastro-intestinal parasite infection, fecal testosterone and facial red coloration. Glucocorticoid levels did not vary systematically with dominance rank, but increased when the dominance hierarchy was unstable, and increased in the presence of receptive females. The relationship between dominance rank and glucocorticoid levels changed direction according to the stability of the dominance hierarchy: glucocorticoid levels were higher in subordinate males under stable conditions, but under conditions of instability higher ranking males had higher glucocorticoid levels. The influence of dominance rank also interacted with the presence of receptive females: glucocorticoids were higher in dominant males than in subordinates, but only during mating periods, suggesting that dominant males are more stressed than subordinates during such periods. These findings support previous studies showing that the relationship between glucocorticoids and dominance rank in male baboons is dependent on the social environment. We also found that males with higher glucocorticoids suffered a higher diversity of gastrointestinal parasite infection, in line with evidence that glucocorticoids suppress the immune system in other species. However, we found no support for the stress-mediated immunocompetence handicap hypothesis for the evolution of condition-dependent ornaments: glucocorticoid and testosterone levels were positively related, rather than the negative relationship predicted by the hypothesis, and we found no relationship between red colour and glucocorticoid levels, suggesting that glucocorticoids do not play a role in translating social conditions or physical health into ornament expression in this species.


Changes in 15NO3 - Availability and Transpiration Rate Are Associated With a Rapid Diurnal Adjustment of Anion Contents as Well as 15N and Water Fluxes Between the Roots and Shoots.
Background and Aims: Understanding interactions between water and nitrate fluxes in response to nitrate availability and transpiration rate is crucial to select more efficient plants for the use of water and nitrate. Methods: Some of these interactions were investigated in intact Brassica napus plants by combining a non-destructive gravimetric device with 15NO3 - labeling. The set-up allowed high-resolution measurement of the effects of a cross-combination of two concentrations of KNO3 or KCl (0.5 and 5 mM) with two different rates of transpiration controlled by the relative humidity during a day-night cycle. Key Results: Results show that (1) high external nitrate concentrations increased root water uptake significantly whatever the transpiration rate, (2) nitrate translocation depended both on the rate of nitrate uptake and loading into xylem (3) dilution-concentration effect of nitrate in the xylem was mainly modulated by both external nitrate availability and transpiration rate, (4) dynamic changes in 15N translocation in the xylem modified shoot growth and capacitance, and (5) variations in tissue concentrations of NO3 - induced by the experimental conditions were balanced by changes in concentrations of chloride and sulfate ions. These effects were even more amplified under low transpiration condition and 0.5 mM external nitrate concentration. Conclusion: Taken together, these results highlight the fine and rapid adjustment of anion contents, nitrate and water flows to changes in transpiration rate and nitrate availability during a day-night cycle. The use of this non-invasive gravimetric device is therefore a powerful tool to assess candidates genes involved in nitrogen and water use efficiency.


Essential regulation of CNS angiogenesis by the orphan G protein-coupled receptor GPR124.
The orphan G protein-coupled receptor (GPCR) GPR124/tumor endothelial marker 5 is highly expressed in central nervous system (CNS) endothelium. Here, we show that complete null or endothelial-specific GPR124 deletion resulted in embryonic lethality from CNS-specific angiogenesis arrest in forebrain and neural tube. Conversely, GPR124 overexpression throughout all adult vascular beds produced CNS-specific hyperproliferative vascular malformations. In vivo, GPR124 functioned cell-autonomously in endothelium to regulate sprouting, migration, and developmental expression of the blood-brain barrier marker Glut1, whereas in vitro, GPR124 mediated Cdc42-dependent directional migration to forebrain-derived, vascular endothelial growth factor-independent cues. Our results demonstrate CNS-specific angiogenesis regulation by an endothelial receptor and illuminate functions of the poorly understood adhesion GPCR subfamily. Further, the functional tropism of GPR124 marks this receptor as a therapeutic target for CNS-related vascular pathologies.


Genetic evidence for functional dependency of p18Ink4c on Cdk4.
The INK4 family of cyclin-dependent kinase (CDK) inhibitors negatively regulates cyclin D-dependent CDK4 and CDK6 and induces the growth-suppressive function of Rb family proteins. Mutations in the Cdk4 gene conferring INK4 resistance are associated with familial and sporadic melanoma in humans and result in a wide spectrum of tumors in mice, suggesting that INK4 is a major regulator of CDK4. Mice lacking the Cdk4 gene exhibit various defects in many organs associated with hypocellularity, whereas loss of the p18(Ink4c) gene results in widespread hyperplasia and organomegaly. To genetically test the notion that the function of INK4 is dependent on CDK4, we generated p18; Cdk4 double-mutant mice and examined the organs and tissues which developed abnormalities when either gene is deleted. We show here that, in all organs we have examined, including pituitary, testis, pancreas, kidney, and adrenal gland, hyperproliferative phenotypes associated with p18 loss were canceled. The double-mutant mice exhibited phenotypes very close to or indistinguishable from that of Cdk4 single-mutant mice. Mice lacking p27(Kip1) develop widespread hyperplasia and organomegaly similar to those developed by p18-deficient mice. The p27; Cdk4 double-mutant mice, however, displayed phenotypes intermediate between those of p27 and Cdk4 single-mutant mice. These results provide genetic evidence that in mice p18(Ink4c) and p27(Kip1) mediate the transduction of different cell growth and proliferation signals to CDK4 and that p18(Ink4c) is functionally dependent on CDK4.


Targeted activation of endothelin-1 exacerbates hypoxia-induced pulmonary hypertension.
Pulmonary arterial hypertension (PAH) is a fatal disease that eventually results in right heart failure and death. Current pharmacologic therapies for PAH are limited, and there are no drugs that could completely cure PAH. Enhanced activity of endothelin system has been implicated in PAH severity and endothelin receptor antagonists have been used clinically to treat PAH. However, there is limited experimental evidence on the direct role of enhanced endothelin system activity in PAH. Here, we investigated the correlation between endothelin-1 (ET-1) and PAH using ET-1 transgenic (ETTG) mice. Exposure to chronic hypoxia increased right ventricular pressure and pulmonary arterial wall thickness in ETTG mice compared to those in wild type mice. Of note, ETTG mice exhibited modest but significant increase in right ventricular pressure and vessel wall thickness relative to wild type mice even under normoxic conditions. To induce severe PAH, we administered SU5416, a vascular endothelial growth factor receptor inhibitor, combined with exposure to chronic hypoxia. Treatment with SU5416 modestly aggravated hypoxia-induced pulmonary hypertension, right ventricular hypertrophy, and pulmonary arterial vessel wall thickening in ETTG mice in association with increased interleukin-6 expression in blood vessels. However, there was no sign of obliterative endothelial cell proliferation and plexiform lesion formation in the lungs. These results demonstrated that enhanced endothelin system activity could be a causative factor in the development of PAH and provided rationale for the inhibition of endothelin system to treat PAH.


The paradoxical role of emotional intensity in the perception of vocal affect.
Vocalizations including laughter, cries, moans, or screams constitute a potent source of information about the affective states of others. It is typically conjectured that the higher the intensity of the expressed emotion, the better the classification of affective information. However, attempts to map the relation between affective intensity and inferred meaning are controversial. Based on a newly developed stimulus database of carefully validated non-speech expressions ranging across the entire intensity spectrum from low to peak, we show that the intuition is false. Based on three experiments (N = 90), we demonstrate that intensity in fact has a paradoxical role. Participants were asked to rate and classify the authenticity, intensity and emotion, as well as valence and arousal of the wide range of vocalizations. Listeners are clearly able to infer expressed intensity and arousal; in contrast, and surprisingly, emotion category and valence have a perceptual sweet spot: moderate and strong emotions are clearly categorized, but peak emotions are maximally ambiguous. This finding, which converges with related observations from visual experiments, raises interesting theoretical challenges for the emotion communication literature.


Growth and body composition in children who are picky eaters: a longitudinal view.
Picky eating may be associated with higher risk of being underweight and poor growth over time or conversely, being overweight. Our aim was to investigate if children identified as picky eaters showed differences in height, weight and body composition from their non-picky peers. Picky eaters were identified in the Avon Longitudinal Study of Parents and Children cohort at 3 years of age. Height and weight were measured on seven occasions (age 7-17 years). Body composition was measured on five occasions by dual-energy x-ray absorptiometry (age 9-17 years). Participants were classified as thin/normal/overweight or obese at each age point using body mass index (BMI) classifications. Data were analysed with adjusted multiple regression analysis and mixed-design repeated measures ANOVA. There was a main effect of being a picky child on height and weight (and on BMI and lean mass index (LMI) in boys) (lower in the picky children, all p ≤ 0.044), but not on percentage body fat or fat mass index (and not on BMI and LMI in girls) (all p > 0.2). The mean heights, weights and BMIs of picky eaters were consistently above the 50th centiles of reference growth charts. More than two-thirds of picky eaters were not thin at any age point. However, being a picky eater was predictive of being thin at a few age points. The growth trajectories of children who were picky eaters were reassuring. The prevalence of thinness amongst some picky eaters is notable, suggesting that some children may need specific early identification, intervention and growth surveillance.


Amphotericin B or ketoconazole therapy of fungal infections in neutropenic cancer patients.
Fungal infections in neutropenic cancer patients have increased in frequency and constitute an important cause of morbidity and mortality. Empiric antifungal therapy is often administered to those patients who have failed to respond to antibacterial antibiotics. We conducted a prospective, randomized trial of amphotericin B and ketoconazole for 172 neutropenic cancer patients with presumed or proven fungal infections. Overall, amphotericin B and ketoconazole were equally effective. Amphotericin B may have been more effective than ketoconazole for the treatment of pneumonia. Also, five of eight Candida tropicalis infections treated with amphotericin B responded, whereas all eight infections treated with ketoconazole failed to respond (P = 0.03). Response rates for localized fungal infections were similar with both drugs. Ketoconazole should not be used as empiric antifungal therapy at institutions where there is a high frequency of infections caused by Aspergillus spp. or C. tropicalis because this agent lacks activity in vitro against these species.


Synergic TiO2 photocatalysis and guanine photoreduction for silver deposition amplification: an ultrasensitive and high-throughput visualized colorimetric analysis strategy for anthrax DNAs in blood using a wettable microwells array.
An ultrasensitive and high-throughput visualized colorimetric method has been initially developed with a wettable microwells array for probing guanine base-containing anthrax DNAs in blood based on silver deposition amplified by the synergic TiO2 photocatalysis and guanine photoreduction under visible light. Hydrophilic microwells were first created on the hydrophobic slides to yield the wettable microwells array, on which photocatalytic titanium dioxide (TiO2) nanoparticles were deposited with dopamine (DA) to yield TiO2@DA for anchoring single strand DNA (ssDNA) capture probes without guanine bases. After the hybridization of the targeted anthrax DNAs, exonuclease I (Exo I) was introduced into the microwells to selectively digest the unhybridized ssDNA probes. The silver deposition was further conducted by the synergic photocatalysis of TiO2@DA and photoreduction of guanine bases of anthrax DNAs, thus achieving the amplified silver signals for the visualized colorimetric assays. Moreover, benefitting from the wettability feature of the hydrophilic-hydrophobic interfaces of the microwells array so fabricated, DNA analytes could be accumulated from the sample droplets through the condensing enrichment process to realize the ultrasensitive detection, in addition to circumventing any crossover contaminants between the sample droplets. The developed visualized colorimetric method with the microwells array was subsequently applied for probing anthrax DNAs in blood with levels down to 1.0 fM. DNAs with single-base and double-base mutations could also be identified accurately. Importantly, such a biosensing design route of a wettable microwells array, in combination with the photocatalytic silver deposition and specific Exo I-catalytic probe digestion, may promise extensive applications for the high-throughput, ultrasensitive, and selective detection of guanine-containing DNA targets with ultra-trace levels in complicated samples like blood.


Survey of ankle-brachial pressure index use and its perceived barriers by general practitioners in the UK.
Peripheral arterial disease (PAD) is often undetected until complications arise, despite it being a major healthcare burden and an independent risk factor for cardiovascular death and systemic atherosclerosis. Appropriate diagnostic tools are as important as clinical knowledge and skill to investigate patients for PAD. Currently, the ankle-brachial pressure index (ABPI) is the recommended diagnostic tool for PAD. We explore current opinions on ABPI by general practitioners (GPs) and the limitations to its implementation in primary care practice. GPs attending a regional 1-day study event, were surveyed in October 2014. Survey questionnaires were placed at the top of each conference pack for each attendee. The survey questionnaire was modelled from the ankle-brachial index (ABI) usage survey questionnaire used in the PAD Awareness, Risk and Treatment: New Resources for Survival (PARTNERS) preceptorship study. All respondents were GPs, with a survey response rate of 77.1%. All respondents regarded ABPI as an important test, that is primarily performed by nursing staff (79.5%) in their respective GP surgeries. 70% and 97% of GPs found ABPI useful for the diagnosis of asymptomatic and symptomatic PAD, respectively. 69% of GPs regarded ABPI as a feasible test in primary care practice. Time constraints (84%), staff availability (89%) and staff training (72%) were cited as the main limitations to its use. Targeted training of nursing staff may improve ABPI usage, although a less time-consuming test for PAD may be another option.


Random versus maximum entropy models of neural population activity.
The principle of maximum entropy provides a useful method for inferring statistical mechanics models from observations in correlated systems, and is widely used in a variety of fields where accurate data are available. While the assumptions underlying maximum entropy are intuitive and appealing, its adequacy for describing complex empirical data has been little studied in comparison to alternative approaches. Here, data from the collective spiking activity of retinal neurons is reanalyzed. The accuracy of the maximum entropy distribution constrained by mean firing rates and pairwise correlations is compared to a random ensemble of distributions constrained by the same observables. For most of the tested networks, maximum entropy approximates the true distribution better than the typical or mean distribution from that ensemble. This advantage improves with population size, with groups as small as eight being almost always better described by maximum entropy. Failure of maximum entropy to outperform random models is found to be associated with strong correlations in the population.


Ursolic acid elicits intrinsic apoptotic machinery by downregulating the phosphorylation of AKT/BAD signaling in human cisplatin‑resistant oral cancer CAR cells.
Oral squamous cell carcinoma (OSCC) is a type of cancer with high morbidity and mortality rates worldwide; it also demonstrates chemotherapeutic resistance. Triterpenoid ursolic acid has been shown to exhibit various biological activities and anticancer effects in several preclinical studies. In our previous study, human cisplatin‑resistant oral cancer CAR cells were established, and the present study aimed to further examine the effects of ursolic acid on CAR cells. The results revealed that ursolic acid inhibited CAR cell viability, as determined using a 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay. Ursolic acid‑induced cell death was mediated through a caspase‑dependent pathway, determined with the pan‑caspase inhibitor, z‑VAD‑fmk. Ursolic acid also increased the activities of caspase‑3 and caspase‑9 in CAR cells, determined by a colorimetric assay. Specifically, the production of reactive oxygen species and loss of mitochondrial membrane potential, detected by flow cytometry, were observed in the ursolic acid‑treated CAR cells. The apoptosis‑associated signaling showed that ursolic acid decreased the phosphorylation of AKT (Ser473) and B‑cell lymphoma 2 (Bcl‑2)‑associated agonist of cell death (BAD; Ser136), and the protein levels of Bcl‑2 and Bcl‑extra large (Bcl‑xL), and increased the expression of BAD and Bcl‑2‑associated X (Bax) protein in CAR cells. In summary, the results supported the potential application of ursolic acid against drug‑resistant oral carcinoma and to improve oral anticancer efficacy in the near future.


Rapid decrease of intact parathyroid hormone could be a predictor of better response to cinacalcet in hemodialysis patients.
Cinacalcet is effective for treating refractory secondary hyperparathyroidism (SHPT), but little is known about the response rates and clinical factors influencing the response. A prospective, single-arm, multi-center study was performed for 24 weeks. Cinacalcet was administered to patients with intact parathyroid hormone (iPTH) level greater than 300 pg/mL. Cinacalcet was started at a dose of 25 mg daily and titrated until 100 mg to achieve a serum iPTH level<300 pg/mL (primary end point). Early response to cinacalcet was defined as a decrease of iPTH more than 50% within one month. Fifty-seven patients were examined. Based on the magnitude of iPTH decrease, patients were divided into responder (n=47, 82.5%) and non-responder (n=10, 17.5%) groups. Among the responders, 38 achieved the primary end point, whereas 9 patients showed a reduction in serum iPTH of 30% or more, but did not reach the primary end point. Compared to non-responders, responders were significantly older (p=0.026), female (p=0.041), and diabetics (p<0.001). Additionally, early response was observed more frequently in the responders (30/47, 63.8%), of whom the majority (27/30, 90.0%) achieved the primary end point. Multivariate analysis showed that lower baseline iPTH levels [odds ratio (OR) 0.96, 95% confidence interval (CI) 0.93-0.99], the presence of diabetes (OR 46.45, CI 1.92-1125.6) and early response (OR 21.54, CI 2.94-157.7) were significant clinical factors affecting achievement of iPTH target. Cinacalcet was effective in most hemodialysis patients with refractory SHPT. The presence of an early response was closely associated with the achievement of target levels of iPTH.


The Effect of Docosahexaenoic Acid and α-Lipoic Acid as Prevention of Bortezomib-Related Neurotoxicity in Patients With Multiple Myeloma.
Background and Aims: In cancer patients, a common complication during chemotherapy is chemotherapy-induced peripheral neuropathy (CIPN). For this reason, we decided to conduct a phase II prospective study on 33 patients with multiple myeloma at first diagnosis, to evaluate whether a nutraceutical compound given for 6 months during bortezomib (BTZ) treatment succeeded in preventing the onset of neurotoxicity. Methods: Neurological evaluation, electroneurography, and functional and quality of life (QoL) scales were performed at baseline and after 6 months. We administered a tablet containing docosahexaenoic acid 400 mg, α-lipoic acid 600 mg, vitamin C 60 mg, and vitamin E 10 mg bid for 6 months. Results: Concerning the 25 patients who completed the study, at 6-month follow-up, 10 patients had no neurotoxicity (NCI-CTCAE [National Cancer Institute-Common Terminology Criteria for Adverse Events] = 0), while 13 progressed to NCI-CTCAE grade 1, 1 had NCI-CTCAE grade 1 with pain, and 1 experienced a NCI-CTCAE grade 2. Painful symptoms were reported only in 2 patients, and we observed stability on functional and QoL scales in all patients. None of the 25 patients stopped chemotherapy due to neurotoxicity. Conclusions: Our data seem to indicate that the co-administration of a neuroprotective agent during BTZ treatment can prevent the appearance/worsening of symptoms related to CIPN, avoiding the interruption of BTZ and maintaining valuable functional autonomy to allow normal daily activities. We believe that prevention remains the mainstay to preserve QoL in this particular patient population, and that future studies with a larger patient population are needed.


Succeed escape: Flow shear promotes tumbling of Escherichia colinear a solid surface.
Understanding how bacteria move close to a surface under various stimuli is crucial for a broad range of microbial processes including biofilm formation, bacterial transport and migration. While prior studies focus on interactions between single stimulus and bacterial suspension, we emphasize on compounding effects of flow shear and solid surfaces on bacterial motility, especially reorientation and tumble. We have applied microfluidics and digital holographic microscopy to capture a large number (>105) of 3D Escherichia coli trajectories near a surface under various flow shear. We find that near-surface flow shear promotes cell reorientation and mitigates the tumble suppression and re-orientation confinement found in a quiescent flow, and consequently enhances surface normal bacterial dispersion. Conditional sampling suggests that two complimentary hydrodynamic mechanisms, Jeffrey Orbit and shear-induced flagella unbundling, are responsible for the enhancement in bacterial tumble motility. These findings imply that flow shear may mitigate cell trapping and prevent biofilm initiation.


The thermally induced decarboxylation mechanism of a mixed-oxidation state carboxylate-based iron metal-organic framework.
Investigations into a thermally generated decarboxylation mechanism for metal site activation and the generation of mesopores in a carboxylate iron-based MOF, PCN-250, have been conducted. PCN-250 exhibits an interesting oxidation state change during thermal treatment under inert atmospheres or vacuum conditions, transitioning from an Fe(iii)3 cluster to a Fe(ii)Fe(iii)2 cluster. To probe this redox event and discern a mechanism of activation, a combination of thermogravimetric analysis, gas sorption, scanning electron microscopy, 57Fe Mössbauer spectroscopy, gas chromatography-mass spectrometry, and X-ray diffraction studies were conducted. The results suggest that the iron-site activation occurs due to ligand decarboxylation above 200 °C. This is also consistent with the generation of a missing cluster mesoporous defect in the framework. The resulting mesoporous PCN-250 maintains high thermal stability, preserving crystallinity after multiple consecutive high-temperature regeneration cycles. Additionally, the thermally reduced PCN-250 shows improvements in the total uptake capacity of methane and CO2.


The role of Cdk5 in retinoic acid-induced apoptosis of cervical cancer cell line.
Cdk5 is a small serine/threonine protein kinase which belongs to Cdk family. Unlike other Cdk members, so far Cdk5 is known to be irrelevant in cell cycle. Cdk5 kinase activity is regulated by binding with its activator, p35. Our previous results indicate that CdkS and p35 are involved in drugs-induced apoptosis of prostate cancer cells. Retinoic acid (RA) is one of the vitamin A-related compounds. Because of its potency on biological functions, it has been widely studied in its novel actions including the ability to inhibit cancer cell growth and to induce apoptosis. Here, we report that RA treatment decreased the growth of human cervical cancer cell line, HeLa, and Cdk5 contributed to this effect. The involvement of Cdk5 in RA-reduced cell survival was performed by treatments of Cdk5 inhibitor and siRNA. We further identified that RA-induced growth inhibition was partly correlated to Cdk5 activity-related apoptosis by detecting cell cycle distribution of sub G1 phase and the signals of Annexin V staining. In addition, our results also indicated that Cdk5 activity was involved in RA-induced HeLa apoptosis by detecting cleavages of caspase-3 and its substrate, PARP (poly (ADP-ribose) polymerases) Interestingly, the nuclear localizations of Cdk5 and p35 proteins were increased by RA treatment, which, again, suggests the involvement of Cdk5 and p35 in RA-induced apoptotic effects. In conclusion, we provide evidence to suggest that Cdk5 and p35 might play important roles in RA-induced HeLa apoptosis.


Holography: an interpretation from the phase-space point of view.
The formation of holograms is interpreted as the consequence of the bilinearity of the ambiguity function. Reconstruction can then be regarded as the manipulation of the ambiguity function. Specifically, we show that in the case of in-line holography, the reconstruction can be regarded as phase tomography. In this way we provide a unified picture for the formulation of both noninterferometric and interferometric phase-retrieval techniques.


Role of TRPV1 and intracellular Ca2+ in excitation of cardiac sensory neurons by bradykinin.
Bradykinin is an important mediator produced during myocardial ischemia and infarction that can activate and/or sensitize cardiac spinal (sympathetic) sensory neurons to trigger chest pain. Because a long-onset latency is associated with the bradykinin effect on cardiac spinal afferents, a cascade of intracellular signaling events is likely involved in the action of bradykinin on cardiac nociceptors. In this study, we determined the signal transduction mechanisms involved in bradykinin stimulation of cardiac nociceptors. Cardiac dorsal root ganglion (DRG) neurons in rats were labeled by intracardiac injection of a fluorescent tracer, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine percholate (DiI). Whole cell current-clamp recordings were performed in acutely isolated DRG neurons. In DiI-labeled DRG neurons, 1 microM bradykinin significantly increased the firing frequency and lowered the membrane potential. Iodoresiniferatoxin, a highly specific transient receptor potential vanilloid type 1 (TRPV1) antagonist, significantly reduced the excitatory effect of bradykinin. Furthermore, the stimulating effect of bradykinin on DiI-labeled DRG neurons was significantly attenuated by baicalein (a selective inhibitor of 12-lipoxygenase) or 2-aminoethyl diphenylborinate [an inositol 1,4,5-trisphosphate (IP(3)) antagonist]. In addition, the effect of bradykinin on cardiac DRG neurons was abolished after the neurons were treated with BAPTA-AM or thapsigargin (to deplete intracellular Ca(2+) stores) but not in the Ca(2+)-free extracellular solution. Collectively, these findings provide new evidence that 12-lipoxygenase products, IP(3), and TRPV1 channels contribute importantly to excitation of cardiac nociceptors by bradykinin. Activation of TRPV1 and the increase in the intracellular Ca(2+) are critically involved in activation/sensitization of cardiac nociceptors by bradykinin.


A Preliminary Comparison of LiverBeads™ with a Conventional Rat Hepatocyte Culture Preparation: Some Aspects of Xenobiotic Metabolism and Related Toxicity.
LiverBeads™ are made of hepatocytes that are immobilised and cryopreserved in alginate gel. They have great potential as an easily transportable and easily handled source of hepatocytes for use in in vitro pharmacotoxicology. In this study, we compared the drug metabolising capacity of LiverBeads in culture with that of conventional cultures and of cultures derived from cryopreserved cells. Trypan blue exclusion, lactate dehydrogenase and DNA content were measured in LiverBead cultures. The levels were all similar to those of the conventional cultures, as were the toxicities of precocene II and allyl alcohol, although more variability was seen in the LiverBeads than in the conventional cultures. The cytochrome P450-dependent activity 3,4-dimethyl-7-ethoxycoumarin-O-dealkylase, was reduced in the LiverBeads when compared to the conventional cultures, although the pattern of conjugation was very similar. In addition, the inducibility of cytochrome P4504A was demonstrated in LiverBeads. Cultures from cryopreserved cells were more susceptible to the toxicants tested, and contained less lactate dehydrogenase and DNA than the conventional cultures. Overall, in terms of the aspects of drug metabolism measured, the cultures from LiverBeads appeared to be equivalent to conventional cultures, and superior to cultures from cryopreserved cells.


Global Kinetic Analysis of Mammalian E3 Reveals pH-dependent NAD+/NADH Regulation, Physiological Kinetic Reversibility, and Catalytic Optimum.
Mammalian E3 is an essential mitochondrial enzyme responsible for catalyzing the terminal reaction in the oxidative catabolism of several metabolites. E3 is a key regulator of metabolic fuel selection as a component of the pyruvate dehydrogenase complex (PDHc). E3 regulates PDHc activity by altering the affinity of pyruvate dehydrogenase kinase, an inhibitor of the enzyme complex, through changes in reduction and acetylation state of lipoamide moieties set by the NAD(+)/NADH ratio. Thus, an accurate kinetic model of E3 is needed to predict overall mammalian PDHc activity. Here, we have combined numerous literature data sets and new equilibrium spectroscopic experiments with a multitude of independently collected forward and reverse steady-state kinetic assays using pig heart E3. The latter kinetic assays demonstrate a pH-dependent transition of NAD(+) activation to inhibition, shown here, to our knowledge, for the first time in a single consistent data set. Experimental data were analyzed to yield a thermodynamically constrained four-redox-state model of E3 that simulates pH-dependent activation/inhibition and active site redox states for various conditions. The developed model was used to determine substrate/product conditions that give maximal E3 rates and show that, due to non-Michaelis-Menten behavior, the maximal flux is different compared with the classically defined kcat.


Pcf11 orchestrates transcription termination pathways in yeast.
In Saccharomyces cerevisiae, short noncoding RNA (ncRNA) generated by RNA polymerase II (Pol II) are terminated by the NRD complex consisting of Nrd1, Nab3, and Sen1. We now show that Pcf11, a component of the cleavage and polyadenylation complex (CPAC), is also generally required for NRD-dependent transcription termination through the action of its C-terminal domain (CTD)-interacting domain (CID). Pcf11 localizes downstream from Nrd1 on NRD terminators, and its recruitment depends on Nrd1. Furthermore, mutation of the Pcf11 CID results in Nrd1 retention on chromatin, delayed degradation of ncRNA, and restricted Pol II CTD Ser2 phosphorylation and Sen1-Pol II interaction. Finally, the pcf11-13 and sen1-1 mutant phenotypes are very similar, as both accumulate RNA:DNA hybrids and display Pol II pausing downstream from NRD terminators. We predict a mechanism by which the exchange of Nrd1 and Pcf11 on chromatin facilitates Pol II pausing and CTD Ser2-P phosphorylation. This in turn promotes Sen1 activity that is required for NRD-dependent transcription termination in vivo.


Progressive strength training in older patients after hip fracture: a randomised controlled trial.
the aim of this study was to assess the effect of a 3-month strength-training programme on functional performance and self-rated health in a group of home-dwelling older hip fracture patients. randomised, controlled; single-blind parallel-group trial. intervention at outpatient's clinic. one hundred and fifty patients with surgical fixation for a hip fracture. strength training was integrated into all stages of the programme. The programme comprised four exercises, half of them in a standing position, performed at 80% of maximum. Measurements were taken after the 3-month intervention. The primary outcome measurement was the Berg Balance Scale (BBS). Secondary outcomes were results of the sit-to-stand test, Timed Up-and-Go test, maximal gait speed, 6-min walk test, Nottingham Extended Activities of Daily Living scale and the SF-12 health status questionnaire. at baseline, there were no significant between-group differences. At follow-up, the intervention group showed highly significant improvements both in the primary endpoint (BBS, mean difference 4.7 points) and in secondary endpoints of tapping strength, mobility and instrumental activities of daily living. home-dwelling hip fracture patients can benefit from an extended supervised strength-training programme in a rehabilitation setting. These patients are capable of high-intensity strength training, which should optimise gains in physical function, strength and balance. Resistance exercise training seems to influence functional performance adaptation.


Curcumin Enhances the Effects of 5-Fluorouracil and Oxaliplatin in Inducing Gastric Cancer Cell Apoptosis Both In Vitro and In Vivo.
Despite the efficacy of fluoropyrimidines and oxaliplatin-based chemotherapy for patients, this treatment leads to significant patient inconvenience, toxicity, and cost. This study aims to validate a nontoxic agent, curcumin, to the current chemotherapeutic regimen. In in vitro experiments, curcumin induced apoptosis in gastric cancer cell line BGC-823. Synergistic antitumor effects of curcumin were observed in combination with 5-fluorouracil (5-FU) and oxaliplatin. These effects were accompanied by downregulation of the expression of Bcl-2 protein and mRNA and upregulation of the expression of Bax and caspase 3, 8, and 9. In addition, the in vivo study showed that the combination of curcumin and 5-FU/oxaliplatin exhibited potent growth inhibition of BGC-823 xenograft tumors. Furthermore, compared with the control group, no significant difference was observed in the body weight of curcumin-treated nude mice. In conclusion, curcumin may act synergistically with the chemotherapeutic regimen FOLFOX in gastric cancer in vitro and in vivo by inducing apoptosis via Bcl/Bax-caspase 8,9-caspase 3 pathway.


The accuracy of distance measurements in solid-state NMR.
The accuracy with which distances can be measured using dipolar recoupling experiments in solid-state NMR is investigated. The relative precision of experiments in a three spin system versus an isolated spin pair is found to depend very strongly on the nature of the coupling Hamiltonian. The accuracy of distances measured in even the simplified three spin system is seen to be very poor for existing homonuclear recoupling Hamiltonians. This suggests that it would be difficult to exploit broadband homonuclear recoupling to measure geometrical information reliably in complex spin systems. These conclusions apply equally to both single-crystal studies and powder samples. In contrast, the presence of additional spins has marginal impact on the accuracy when the coupling Hamiltonians commute with each other, as in the case of heteronuclear recoupling. The possibility of creating such a Hamiltonian for homonuclear recoupling using a suitable rotor-synchronized pulse sequence is discussed.


Optimal timing of ureteroscopic lithotripsy after the initial drainage treatment and risk factors for postoperative febrile urinary tract infection in patients with obstructive pyelonephritis: a retrospective study.
A history of preoperative obstructive pyelonephritis has been reported as a risk factor for febrile urinary tract infection (fUTI) after ureteroscopic lithotripsy (URSL). But there is no clear evidence of risk factors for developing fUTI including the optimal timing of URSL after obstructive pyelonephritis treatment. Of the 1361 patients, who underwent URSL at our hospital from January 2011 to December 2017, 239 patients had a history of pre-URSL obstructive pyelonephritis. The risk factors were analyzed by comparing the patients' backgrounds with the presence or absence of fUTI after URSL. The factors examined were age, gender, body mass index, comorbidity, presence or absence of preoperative ureteral stent, stone position, stone laterality, stone size, Hounsfield unit (HU) value on computed tomography scan, history of sepsis during obstructive pyelonephritis, period from antipyresis to URSL, ureteral stenting period, operation time, and presence or absence of access sheath at URSL. In addition, the stone components and renal pelvic urinary culture bacterial species during pre-URSL pyelonephritis were also examined. Post-URSL fUTI developed in 32 of 239 patients (13.4%), and 11 of these 32 cases led to sepsis (34.4%). Univariate analysis showed that stone position, stone maximum HU value, presence of sepsis during obstructive pyelonephritis, period from antipyresis to URSL, pre-URSL ureteral stent placement, operation time were risk factors of fUTI. Stone components and urinary cultures during pyelonephritis were not associated with risk of fUTI. Multivariate analysis showed that renal stone position, pre-URSL ureteral stent placement > 21 days, and operation time > 75 min were independent risk factors of fUTI following the URSL. F-UTI following the URSL could be avoided by ureteral stent placement period 21 days or less and operation time 75 min or less in patients with obstructive pyelonephritis.


Somatic embryogenesis and enhanced shoot organogenesis in Metabriggsia ovalifolia W. T. Wang.
An efficient protocol providing a dual regeneration pathway via direct shoot organogenesis and somatic embryogenesis for an endangered species, Metabriggsia ovalifolia W. T. Wang, was established from leaf explants. When applied at 2.5 μM, the cytokinins 6-benzyladenine (BA) or thidiazuron (TDZ) and the auxins indole-3-butyric acid (IBA), α-naphthaleneacetic acid (NAA) and indole-3-acetic acid (IAA) could induce shoots when on basal Murashige and Skoog (MS) medium. BA and TDZ could induce more adventitious shoots (19.1 and 31.2/explant, respectively) than NAA (4.6/explant), IBA (5.7/explant) or IAA (6.4/explant). BA and TDZ at 5-10 μM could induce both shoots and somatic embryos. A higher concentration of TDZ (25 μM) induced only somatic embryos (39.8/explant). The same concentration of BA induced both adventitious shoots (23.6/explant) and somatic embryos (9.7/explant). Thus, somatic embryogenesis in this plant needs a high cytokinin concentration (BA; TDZ), as evidenced by histology. Somatic embryos germinated easily when left on the same media, but formed adventitious roots in two weeks on MS supplemented with 0.5 μM NAA, 0.5 μM IBA and 0.1% activated charcoal. Over 93% of plantlets survived following acclimatization and transfer to a mixture of sand and vermiculite (1:1, v/v) in trays.


Bone calcification and calcium homeostasis in rats with non-insulin-dependent diabetes induced by streptozocin.
The effect of mild, non-insulin-dependent diabetes (NIDDM) on bone calcification and calcium (Ca) homeostasis was studied in growing rats (males and females). The diabetic state was characterized by mild insulin deficiency, plasma levels being 73% of controls, and mild hyperglycemia, with nonfasting plasma glucose levels of 1.5 times normal. There was no difference in plasma levels of Ca, phosphate (Pi), magnesium (Mg), alkaline phosphatase, immunoreactive parathyroid hormone (iPTH), calcitonin, 25-(OH)vitamin D (25[OH]D), 1,25-dihydroxyvitamin D (1,25[OH]2D), and 24,25-dihydroxyvitamin D (24,25[OH]2D) between the NIDDM rats and their controls of either sex. Metabolic Ca and Pi balance studies revealed that the experimental animals of both sexes were in positive Ca and Pi balance similar to that of their controls. Histologic studies of the kidney and intestinal slices from the experimental group were normal. Ca and Pi bone content calculated per gram bone ash of the femur, mandible, and second and fourth caudal vertebrae, and the organic content in the bones of the NIDDM animals showed no difference from their controls. Femur bone density and tibial epiphyseal growth plate width and morphology were similar histologically in the experimental and control rats. No decreased osteoid content in the tibial bone was found in the diabetic rats compared with controls. Physiologic sex differences, consisting of lower plasma Pi, higher plasma calcitonin levels, increased ratio of femur dry bone weight to total body weight, and increased percentage of mineralized and total bone volume at the tibial metaphysis seen in female compared with male control rats were also seen in the diabetic animals.(ABSTRACT TRUNCATED AT 250 WORDS)


Primary osteosarcoma in patients older than 40 years of age.
Among the 665 patients who registered at our hospital, we reviewed 39 cases of high grade primary osteosarcoma in patients who were older than 40 yr of age. The aim of this study was to determine if a primary osteosarcoma in older patients has different clinical features, and a poorer prognosis than in younger patients. Two evaluations were performed. In the first, an attempt was made to determine the possible prognostic factors such as gender, location, size, alkaline phosphatase, radiological findings, chemotherapy intensity, chemotherapy-induced tumor necrosis, and surgical margin. The second evaluation involved assessment of whether there were any significant clinical differences between older patients and adolescents. According to the results, a primary osteosarcoma in older patients did not reveal any significant prognostic variables. A primary osteosarcoma in older patients showed a poorer prognosis due to relatively unusual locations, common abnormal radiological findings, and a poor response to chemotherapy. Therefore, careful attention should be paid to making an accurate diagnosis and new strategies for more effective treatment, including chemotherapy, must to be developed in order to achieve long term survival in older patients with osteosarcoma.


Effect of FGF/FGFR pathway blocking on lung adenocarcinoma and its cancer-associated fibroblasts.
Cancer-associated fibroblasts (CAFs) are known to promote tumourigenesis through various mechanisms. Fibroblast growth factor (FGF)/FGF receptor (FGFR)-dependent lung cancers have been described. We have developed a mouse model of lung adenocarcinoma that was constructed through the induction of Fgf9 overexpression in type 2 alveolar cells. The expression of Fgf9 in adult lungs resulted in the rapid development of multiple adenocarcinoma-like tumour nodules. Here, we have characterised the contribution of CAFs and the Fgf/Fgfr signalling pathway in maintaining the lung tumours initiated by Fgf9 overexpression. We found that CAF-secreted Fgf2 contributes to tumour cell growth. CAFs overexpressed Tgfb, Mmp7, Fgf9, and Fgf2; synthesised more collagen, and secreted inflammatory cell-recruiting cytokines. CAFs also enhanced the conversion of tumour-associated macrophages (TAMs) to the tumour-supportive M2 phenotype but did not influence angiogenesis. In vivo inhibition of Fgfrs during early lung tumour development resulted in significantly smaller and fewer tumour nodules, whereas inhibition in established lung tumours caused a significant reduction in tumour size and number. Fgfr inhibition also influenced tumour stromal cells, as it significantly abolished TAM recruitment and reduced tumour vascularity. However, the withdrawal of the inhibitor caused a significant recurrence/regrowth of Fgf/Fgfr-independent lung tumours. These recurrent tumours did not possess a higher proliferative or propagative potential. Our results provide evidence that fibroblasts associated with the Fgf9-induced lung adenocarcinoma provide multiple means of support to the tumour. Although the Fgfr blocker significantly suppressed the tumour and its stromal cells, it was not sufficient to completely eliminate the tumour, probably due to the emergence of alternative (resistance/maintenance) mechanism(s). This model represents an excellent tool to further study the complex interactions between CAFs, their related chemokines, and the progression of lung adenocarcinoma; it also provides further evidence to support the need for a combinatorial strategy to treat lung cancer. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


aPKC-mediated phosphorylation regulates asymmetric membrane localization of the cell fate determinant Numb.
In Drosophila, the partition defective (Par) complex containing Par3, Par6 and atypical protein kinase C (aPKC) directs the polarized distribution and unequal segregation of the cell fate determinant Numb during asymmetric cell divisions. Unequal segregation of mammalian Numb has also been observed, but the factors involved are unknown. Here, we identify in vivo phosphorylation sites of mammalian Numb and show that both mammalian and Drosophila Numb interact with, and are substrates for aPKC in vitro. A form of mammalian Numb lacking two protein kinase C (PKC) phosphorylation sites (Numb2A) accumulates at the cell membrane and is refractory to PKC activation. In epithelial cells, mammalian Numb localizes to the basolateral membrane and is excluded from the apical domain, which accumulates aPKC. In contrast, Numb2A is distributed uniformly around the cell cortex. Mutational analysis of conserved aPKC phosphorylation sites in Drosophila Numb suggests that phosphorylation contributes to asymmetric localization of Numb, opposite to aPKC in dividing sensory organ precursor cells. These results suggest a model in which phosphorylation of Numb by aPKC regulates its polarized distribution in epithelial cells as well as during asymmetric cell divisions.


Positive pre-biopsy MRI: are systematic biopsies still useful in addition to targeted biopsies?
The diagnostic strategy implementing multiparametric magnet resonance tomography (mpMRI) and targeted biopsies (TB) improves the detection and characterization of significant prostate cancer (PCa). We aimed to assess the clinical usefulness of systematic biopsies (SB) in the setting of patients having a pre-biopsy positive MRI. A review of the literature was performed in March 2018. All studies investigating the performance of SB in addition to TB (all techniques) were assessed, both in the biopsy-naïve and repeat biopsy setting. Evidence demonstrates that TB improves the detection of index-significant PCa compared with SB alone, in both initial and repeat biopsy settings. However, the combination of both TB and SB improved the overall (around 30%) and significant (around 10%) PCa detection rates as compared with TB alone. Significant differences between both biopsy approaches exist regarding cancer location favoring SB for the far lateral sampling, and TB for the anterior zone. Main current pitfalls of pure TB strategy are the learning curve and experience required for mpMRI reading and biopsy targeting, as well as the precision assessment in TB techniques. A pure TB strategy omitting SB leads to the risk of missing up to 15% of significant cancer, due to limitations of mpMRI performance/reading and of precision during lesion targeting. SB remain necessary, in addition to the TB, to obtain the most accurate assessment of the entire prostate gland in this sub-group of patients at risk of significant disease.


[Differences by age in clinical features of inflammatory bowel disease].
Approximately, 15% of patients with Inflammatory Bowel Disease (IBD) are diagnosed at 60 years of age or more. To characterize and compare clinical variables between patients with IBD aged 60 years or more and their younger counterparts. Retrospective study based on a registry of IBD patients diagnosed between the years 1976 and 2014. Four hundred and nine IBD patients were included. Among them, 294 had Ulcerative Colitis (UC), 104 had Crohn's Disease (CD) and eleven had an indeterminate IBD. Forty-six patients (11.2%) were older than 60 years and 16 (3.9%) had been diagnosed after this age. When comparing patients by age, those aged 60 years or more had a higher frequency of CD and indeterminate IBD (p < 0.01) and a lower ileocolic location in CD (p = 0.02). Both groups were similar in terms of hospitalization due to IBD flare, surgery, use of steroids, immunosuppressive or biological therapies and drug-related adverse events. When analyzing age at diagnosis of IBD, patients diagnosed at ages of 60 years or more had a lower frequency of UC (p < 0.01), a higher frequency of exclusive colonic involvement (p = 0.01), and lower use of mesalamine (p < 0.01). There were no differences in drug-related adverse events, hospitalizations due to IBD flares and surgery according to age at diagnosis. In this population, clinical features of IBD in older patients were similar to those in younger patients.


Assessing Social Contagion in Body Mass Index, Overweight, and Obesity Using a Natural Experiment.
Little is known about whether the substantial clustering of obesity and overweight within social and geographic networks results from causal pathways, such as social contagion and shared environments, or from self-selection. This study aimed to determine whether exposure to communities with higher rates of obesity increases the body mass index (BMI) of individuals, calculated as weight in kilograms divided by height in meters squared, and their risk of being overweight or obese, and whether social contagion, shared environments, or self-selection can account for identified differences. This natural experiment study used the routine assignment of military service members to installations as a source of exogenous variation in exposure to communities with higher vs lower rates of obesity. The study, which used data collected by the Military Teenagers' Environments, Exercise, and Nutrition Study, examined families from 38 military installations around the United States to determine if individuals had higher BMI and greater odds of overweight and obesity when assigned to installations in counties with higher rates of obesity. The study also examined if the relationship persisted after controlling for shared built environments. The participants included 1 parent and 1 child aged 12 or 13 years from 1519 families of Army-enlisted personnel. Data analysis was completed from November 2016 to October 2017. Adult obesity rate in the county where the assigned installation of the service member was located. Time at installation and location of residence (on-installation vs off-installation) were used to measure the degree of exposure. For parents, outcomes were BMI, overweight/obesity (BMI, ≥25) and obesity (BMI, ≥30). For children, outcomes were BMI z score, overweight/obesity (BMI percentile for age and sex, ≥85), and obesity (BMI percentile for age and sex, ≥95). These outcomes were based on self-reports for parents, self-reports and parent reports for all children, and anthropometric measurements for a subsample of children. Members of 1519 families participated, including 1314 adults (of whom 740, or 56%, were fathers) and 1111 children (of whom 576, or 52%, were boys); anthropometric measurements were performed on 458 children. The sample was 40% white, 22% black, 24% Hispanic, and 14% other races/ethnicities. A 1-percentage point higher county obesity rate was associated with a higher BMI (a difference of 0.08; 95% CI, 0.02-0.13) and greater odds of obesity (adjusted odds ratio [aOR], 1.05; 95% CI, 1.02-1.08) in parents, and a higher BMI z score (0.01; 95% CI, 0.003-0.02) and greater odds of overweight/obesity (aOR, 1.04; 95% CI, 1.01-1.06) in children. The evidence supported stronger associations among families with more time at installation and off-installation residence. Associations persisted even after controlling for shared built environments. Exposure to counties with higher rates of obesity was associated with higher BMI and higher odds of overweight and/or obesity in parents and children. There was no evidence to support self-selection or shared built environments as possible explanations, which suggests the presence of social contagion in obesity.


Cryptosporidium as a testbed for single cell genome characterization of unicellular eukaryotes.
Infectious disease involving multiple genetically distinct populations of pathogens is frequently concurrent, but difficult to detect or describe with current routine methodology. Cryptosporidium sp. is a widespread gastrointestinal protozoan of global significance in both animals and humans. It cannot be easily maintained in culture and infections of multiple strains have been reported. To explore the potential use of single cell genomics methodology for revealing genome-level variation in clinical samples from Cryptosporidium-infected hosts, we sorted individual oocysts for subsequent genome amplification and full-genome sequencing. Cells were identified with fluorescent antibodies with an 80 % success rate for the entire single cell genomics workflow, demonstrating that the methodology can be applied directly to purified fecal samples. Ten amplified genomes from sorted single cells were selected for genome sequencing and compared both to the original population and a reference genome in order to evaluate the accuracy and performance of the method. Single cell genome coverage was on average 81 % even with a moderate sequencing effort and by combining the 10 single cell genomes, the full genome was accounted for. By a comparison to the original sample, biological variation could be distinguished and separated from noise introduced in the amplification. As a proof of principle, we have demonstrated the power of applying single cell genomics to dissect infectious disease caused by closely related parasite species or subtypes. The workflow can easily be expanded and adapted to target other protozoans, and potential applications include mapping genome-encoded traits, virulence, pathogenicity, host specificity and resistance at the level of cells as truly meaningful biological units.


Pycnogenol® and Centella asiatica in the management of asymptomatic atherosclerosis progression.
The aim of the study was to evaluate the effect of the nutritional supplements Pycnogenol® and total triterpenic fraction of Centella asiatica (TTFCA) on atherosclerosis progression in low-risk asymptomatic subjects with carotid or femoral stenosing plaques. This was an observational pilot, substudy of the San Valentino epidemiological cardiovascular study. The study included 824 subjects aged 45-60 without any conventional risk factors who had a stenosing atherosclerotic plaque (>50-60%) in at least one carotid or common femoral bifurcation, allocated into 6 groups: Group 1 (Controls): management was based on education, exercise, diet and lifestyle changes. This same management plan was used in all other groups; group 2: Pycnogenol® 50 mg/day; group 3: Pycnogenol® 100 mg/day; group 4: Aspirin® 100 mg/day or ticlopidine 250 mg/day if intolerant to aspirin; group 5: Aspirin® 100 mg/day and Pycnogenol® 100 mg/day; group 6: Pycnogenol® 100 mg/day plus TTFCA 100 mg/day. The follow-up lasted 42 months. Plaque progression was assessed using the ultrasonic arterial score based on the arterial wall morphology and the number of plaques that progressed and on the number of subjects that had cardiovascular events. A secondary endpoint was to evaluate the changes in oxidative stress at baseline and at 42 months. The ultrasonic score increased significantly in groups 1, 2, and 4 (>1%) but not in groups 3, 5 and 6 (<1%) suggesting a beneficial effect of Pycnogenol® 100 mg. Considering the percent of patients that progressed from class V (asymptomatic) to VI (symptomatic) there was a progression of plaques in 48.09% of controls. In the Pycnogenol® 100 (group 3, 10.4%) and in the Aspirin®+ Pycnogenol® (group 5, 10.68%) progression was half of what observed with antiplatelet agent (group 4, 20.93%); in the TTFCA+ Pycnogenol®group (group 6) progression was 7.4 times lower than in controls; 3.22 times lower than in the antiplatelet agents group (4). Events (hospital admission, specialized care) were observed in 16.03% of controls; there were 8.83% of subjects with events with Pycnogenol® 50 mg and 8% in group 3 (Pycnogenol® 100 mg). In group 4 (antiplatelets), 8.52% of subjects had events; in group 5, 6.87% of subjects had events and in group 6 (TTFCA+ Pycnogenol®) only 4.41% had events (this was the lowest event rate; P<0.05). All treatment groups had a significantly lower event rate (P<0.05) in comparison with controls. Considering treatments groups 2, 3, 5, 6 had a lower number (P<0.05) of subjects in need of cardiovascular management in comparison with controls. The need for risk factor management was higher in controls and lower in group 6 (P<0.05). In groups 2 to 6 the need for risk factor management was lower than in controls (P<0.05). Including all events (hospital admission, need for treatment or for risk management) 51.9% of controls were involved. In the other groups there was a reduction (from a -9.28% reduction in group 2 to a -26% in group 6) (P<0.002). The most important reduction (higher that in all groups; P<0.05) was in group 6. At 42 months, oxidative stress in all the Pycnogenol® groups was less than in the control group. In the combined group of Pycnogenol® and TTFCA the oxidative stress was less than with Pycnogenol® alone (P<0.001). Pycnogenol® and the combination of Pycnogenol® +TTFCA appear to reduce the progression of subclinical arterial plaques and the progression to clinical stages. The reduction in plaque and clinical progression was associated with a reduction in oxidative stress. The results justify a large, randomized, controlled study to demonstrate the efficacy of the combined Pycnogenol® and TTFCA prophylactic therapy in preclinical atherosclerosis.


Signet ring cell mesothelioma; A diagnostic challenge.
Signet ring cell mesothelioma is a rare variant of epithelioid mesothelioma with limited cases published. It has a male predilection and most commonly occurs on pleura; it can also arise in the peritoneal cavity. The signet ring cell morphology can pose a challenge leading to a potential diagnostic error. A variety of benign and malignant diseases, including reactive histiocytic hyperplasia, adenocarcinoma, melanoma, and lymphoma with signet ring cell morphology should be considered in the differential diagnosis. In signet ring cell mesothelioma work up, mucin stains are of limited value. Even though immunohistochemistry is routinely used in mesothelioma diagnosis, there is no sole specific mesothelial marker. Hence, a panel of mesothelial and epithelial markers are used; these should be interpreted with caution especially in this variant. Electron microscopy and genetic testing can be very helpful in distinguishing signet ring cell mesothelioma from its mimickers.


Cytokine cascade in sepsis.
Sepsis is associated with an exacerbated production of both pro- and anti-inflammatory cytokines, which are detectable within the bloodstream. Their 'half-angel, half-devil' properties are fully illustrated in sepsis. While they are a prerequisite to fight infection, their overzealous production is deleterious. The highest levels are found in plasma of non-surviving patients: they are markers and causative agents of poor outcome. Only the level of the chemokine RANTES is inversely associated with the APACHE II score (r = -0.7; p = 0.02) and low levels are associated with poor outcome. The link, interplay and network of cytokines taking place during sepsis are illustrated by the correlations between the levels of most pro- and anti-inflammatory cytokines. Excessive release of anti-inflammatory cytokines may be associated with the immunodysregulation observed in sepsis. However, despite the presence of huge amounts of anti-inflammatory cytokines and molecules targeting specifically interleukin-1 (IL-1) (i.e. IL-1 receptor antagonist) and tumour necrosis factor (TNF) (i.e. soluble TNF receptors), there is no indication that their levels are sufficient to counteract fully these proinflammatory cytokines. TNF was initially thought to be the 'hub of the cytokine network'. Although TNF contributes towards favouring the production of many other cytokines within a complex cascade, there are numerous examples to illustrate that its presence is not a prerequisite for these productions.


Suppressing TGFβ signaling in regenerating epithelia in an inflammatory microenvironment is sufficient to cause invasive intestinal cancer.
Genetic alterations in the TGFβ signaling pathway in combination with oncogenic alterations lead to cancer development in the intestines. However, the mechanisms of TGFβ signaling suppression in malignant progression of intestinal tumors have not yet been fully understood. We have examined Apc(Δ716) Tgfbr2(ΔIEC) compound mutant mice that carry mutations in Apc and Tgfbr2 genes in the intestinal epithelial cells. We found inflammatory microenvironment only in the invasive intestinal adenocarcinomas but not in noninvasive benign polyps of the same mice. We thus treated simple Tgfbr2(ΔIEC) mice with dextran sodium sulfate (DSS) that causes ulcerative colitis. Importantly, these Tgfbr2(ΔIEC) mice developed invasive colon cancer associated with chronic inflammation. We also found that TGFβ signaling is suppressed in human colitis-associated colon cancer cells. In the mouse invasive tumors, macrophages infiltrated and expressed MT1-MMP, causing MMP2 activation. These results suggest that inflammatory microenvironment contributes to submucosal invasion of TGFβ signaling-repressed epithelial cells through activation of MMP2. We further found that regeneration was impaired in Tgfbr2(ΔIEC) mice for intestinal mucosa damaged by DSS treatment or X-ray irradiation, resulting in the expansion of undifferentiated epithelial cell population. Moreover, organoids of intestinal epithelial cells cultured from irradiated Tgfbr2(ΔIEC) mice formed "long crypts" in Matrigel, suggesting acquisition of an invasive phenotype into the extracellular matrix. These results, taken together, indicate that a simple genetic alteration in the TGFβ signaling pathway in the inflamed and regenerating intestinal mucosa can cause invasive intestinal tumors. Such a mechanism may play a role in the colon carcinogenesis associated with inflammatory bowel disease in humans.


Type, severity, management and outcome of ocular and adnexal firework-related injuries: the Rotterdam experience.
To study the type, severity, management and outcome of firework-related adnexal and ocular injuries during New Year's Eve festivities. A retrospective analysis of 123 injured patients (143 eyes) treated at the Rotterdam Eye Hospital between 2009 and 2013. All ages were included and analysed according to age, gender, active participant or bystander, laterality, location, dimension and severity of injury. Outcome parameter was the final best-corrected visual acuity. The mean age was 22 ± 13 years with 87% males and 53% bystanders. 52% were ≤18 years. There was a higher number of female than male bystanders (63% versus 51%, p = 0.30). 50% of the eyes sustained mild, 13% moderate and 37% severe trauma. Adults suffered more from severe injuries compared to children (42% versus 31%). The most frequent intervention was gunpowder removal (20%), followed by traumatic cataract surgery (12%) and amniotic membrane grafting (8%). 76% of patients were followed over 1 year. At the end of follow-up, 88 (61.5%) eyes had recovered fully, while 55 (38.5%) eyes suffered from persistent complications with reduced vision ≤0.8 in 30% of injured eyes. 15 patients (12%, 10 adults, five children) were considered legally blind (vision ≤0.1). Three (2%) eyes were subject to evisceration. Every year, around New Year's Eve 30-45 victims were referred to the Rotterdam Eye Hospital; 50% sustained moderate-to-severe trauma. In severe firework injuries, patients required multiple treatments that may not prevent permanent blindness and/or functional/cosmetic disfigurement. The majority was bystander and younger than 18 years.


Murine model of dextran sulfate sodium-induced colitis reveals Candida glabrata virulence and contribution of β-mannosyltransferases.
Candida glabrata, like Candida albicans, is an opportunistic yeast pathogen that has adapted to colonize all segments of the human gastrointestinal tract and vagina. The C. albicans cell wall expresses β-1,2-linked mannosides (β-Mans), promoting its adherence to host cells and tissues. Because β-Mans are also present in C. glabrata, their role in C. glabrata colonization and virulence was investigated in a murine model of dextran sulfate sodium (DSS)-induced colitis. Five clustered genes of C. glabrata encoding β-mannosyltransferases, BMT2-BMT6, were deleted simultaneously. β-Man expression was studied by Western blotting, flow cytometry, and NMR analysis. Mortality, clinical, histologic, and colonization scores were determined in mice receiving DSS and different C. glabrata strains. The results show that C. glabrata bmt2-6 strains had a significant reduction in β-1,2-Man expression and a disappearance of β-1,2-mannobiose in the acid-stable domain. A single gavage of C. glabrata wild-type strain in mice with DSS-induced colitis caused a loss of body weight, colonic inflammation, and mortality. Mice receiving C. glabrata bmt2-6 mutant strains had normal body weight and reduced colonic inflammation. Lower numbers of colonies of C. glabrata bmt2-6 were recovered from stools and different parts of the gastrointestinal tract. Histopathologic examination revealed that the wild-type strain had a greater ability to colonize tissue and cause tissue damage. These results showed that C. glabrata has a high pathogenic potential in DSS-induced colitis, where β-Mans contribute to colonization and virulence.


Frequent chromatin rearrangements in myelodysplastic syndromes--what stands behind?
Myelodysplastic syndromes (MDS) represent a clinically and genetically heterogeneous group of clonal haematopoietic diseases characterized by a short survival and high rate of transformation to acute myeloid leukaemia (AML). In spite of this variability, MDS is associated with typical recurrent non-random cytogenetic defects. Chromosomal abnormalities are detected in the malignant bone-marrow cells of approximately 40-80 % of patients with primary or secondary MDS. The most frequent chromosomal rearrangements involve chromosomes 5, 7 and 8. MDS often shows presence of unbalanced chromosomal changes, especially large deletions [del(5), del(7q), del(12p), del(18q), del(20q)] or losses of whole chromosomes (7 and Y). The most typical cytogenetic abnormality is a partial or complete deletion of 5q- that occurs in roughly 30 % of all MDS cases either as the sole abnormality or in combination with other aberrations as a part of frequently complex karyotypes. The mechanisms responsible for the formation of MDS-associated recurrent translocations and complex karyotypes are unknown. Since some of the mentioned aberrations are characteristic for several haematological malignancies, more general cellular conditions could be expected to play a role. In this article, we introduce the most common rearrangements linked to MDS and discuss the potential role of the non-random higher-order chromatin structure in their formation. A contribution of the chromothripsis - a catastrophic event discovered only recently - is considered to explain how complex karyotypes may occur (during a single event).


Cloning and characterization of an aminoglycoside resistance determinant from Micromonospora zionensis.
The sisomicin-gentamicin resistance methylase (sgm) gene was isolated from Micromonospora zionensis and cloned in Streptomyces lividans. The sgm gene was expressed in Micromonospora melanosporea, where its own promoter was active, and also in Escherichia coli under the control of the lacZ promoter. The complete nucleotide sequence of 1,122 bp and a transcription start point were determined. The sequence contains an open reading frame that encodes a polypeptide of 274 amino acids. The methylation of 30S ribosomal subunits by Sgm methylase accounts adequately for all known resistance characteristics of M. zionensis, but expression of high-level resistance to hygromycin B is background dependent. A comparison of the amino acid sequence of the predicted Sgm protein with the deduced amino acid sequences for the 16S rRNA methylases showed extensive similarity of Grm and significant similarity to KgmB but not to KamB methylase.


Uptake of glucose into the intestine of diabetic rats: effects of variations in the effective resistance of the unstirred water layer.
Diabetes mellitus in rats is associated with enhanced intestinal absorption of glucose, but the kinetic basis for this observation is disputed. Only recently has it been appreciated that failure to account for the resistance of the unstirred water layer (UWL) leads to gross errors in the estimation of the kinetics of active and passive transport. Accordingly, the uptake Jd (unidirectional flux rate) of glucose into the intestine was measured in alloxan- or streptozotocin-diabetic rats (DM) and in control animals (C) under conditions selected to yield low and high UWL resistance. When the UWL resistance was minimized by stirring the bulk phase, Jd was higher in DM than in C, but only from the higher concentrations of glucose; this difference between DM and C was obscured when UWL was high. The difference between C and DM in Jd from 40 mM glucose progressively increased from 3 to 83 days after induction of DM, and was present in the jejunum and ileum as well as in young and old animals. The Jd from 0.5 to 2 mM glucose was similar in DM and C, and the inhibitory effect of galactose on Jd of glucose was similar in DM and C. Jd was higher in DM than in C for L-glucose and for D-glucose in the presence of phlorizin and in the absence of sodium ion. Kinetic analysis demonstrated a higher maximal transport rate (Jdm) and apparent passive permeability coefficient (P*) in DM than in C, but the apparent affinity constant was unchanged. It is concluded that (1) the greater Jd of glucose in DM is due to a greater Jdm and P*, rather than due to changes in the properties of the glucose carrier itself, and (2) these differences may be obscured by variations in UWL.


Emotions and motivated behavior converge on an amygdala-like structure in the zebrafish.
The brain reward circuitry plays a key role in emotional and motivational behaviors, and its dysfunction underlies neuropsychiatric disorders such as schizophrenia, depression and drug addiction. Here, we characterized the neuronal activity pattern induced by acute amphetamine administration and during drug-seeking behavior in the zebrafish, and demonstrate the existence of conserved underlying brain circuitry. Combining quantitative analyses of cfos expression with neuronal subtype-specific markers at single-cell resolution, we show that acute d-amphetamine administration leads to both increased neuronal activation and the recruitment of neurons in the medial (Dm) and the lateral (Dl) domains of the adult zebrafish pallium, which contain homologous structures to the mammalian amygdala and hippocampus, respectively. Calbindin-positive and glutamatergic neurons are recruited in Dm, and glutamatergic and γ-aminobutyric acid (GABAergic) neurons in Dl. The drug-activated neurons in Dm and Dl are born at juvenile stage rather than in the embryo or during adulthood. Furthermore, the same territory in Dm is activated during both drug-seeking approach and light avoidance behavior, while these behaviors do not elicit activation in Dl. These data identify the pallial territories involved in acute psychostimulant response and reward formation in the adult zebrafish. They further suggest an evolutionarily conserved function of amygdala-like structures in positive emotions and motivated behavior in zebrafish and mammals.


Mechanisms of adaptation from a multiple to a single step recovery strategy following repeated exposure to forward loss of balance in older adults.
When released from an initial, static, forward lean angle and instructed to recover with a single step, some older adults are able to meet the task requirements, whereas others either stumble or fall. The purpose of the present study was to use the concept of margin of stability (MoS) to investigate balance recovery responses in the anterior-posterior direction exhibited by older single steppers, multiple steppers and those that are able to adapt from multiple to single steps following exposure to repeated forward loss of balance. One hundred and fifty-one healthy, community dwelling, older adults, aged 65-80 years, participated in the study. Participants performed four trials of the balance recovery task from each of three initial lean angles. Balance recovery responses in the anterior-posterior direction were quantified at three events; cable release (CR), toe-off (TO) and foot contact (FC), for trials performed at the intermediate lean angle. MoS was computed as the anterior-posterior distance between the forward boundary of the Base of Support (BoS) and the vertical projection of the velocity adjusted centre of mass position (XCoM). Approximately one-third of participants adapted from a multiple to a single step recovery strategy following repeated exposure to the task. MoS at FC for the single and multiple step trials in the adaptation group were intermediate between the exclusively single step group and the exclusively multiple step group, with the single step trials having a significant, 3.7 times higher MoS at FC than the multiple step trials. Consistent with differences between single and multiple steppers, adaptation from multiple to single steps was attributed to an increased BoS at FC, a reduced XCoM at FC and an increased rate of BoS displacement from TO to FC. Adaptations occurred within a single test session and suggest older adults that are close to the threshold of successful recovery can rapidly improve dynamic stability following repeated exposure to a forward loss of balance.


Surveillance of cirrhosis for hepatocellular carcinoma: a cost-utility analysis.
Using a decision-analytic model, we evaluated the effectiveness and cost-effectiveness of surveillance for hepatocellular carcinoma (HCC) in individuals with cirrhosis. Separate cohorts with cirrhosis due to alcoholic liver disease, hepatitis B and hepatitis C were simulated. Results were also combined to approximate a mixed aetiology population. Comparisons were made between a variety of surveillance algorithms using alpha-foetoprotein (AFP) assay and/or ultrasound at 6- and 12-monthly intervals. Parameter estimates were obtained from comprehensive literature reviews. Uncertainty was explored using one-way and probabilistic sensitivity analyses. In the mixed aetiology cohort, 6-monthly AFP+ultrasound was predicted to be the most effective strategy. The model estimates that, compared with no surveillance, this strategy may triple the number of people with operable tumours at diagnosis and almost halve the number of people who die from HCC. The cheapest strategy employed triage with annual AFP (incremental cost-effectiveness ratio (ICER): 20,700 pounds per quality-adjusted life-year (QALY) gained). At a willingness-to-pay threshold of 30,000 pounds per QALY the most cost-effective strategy used triage with 6-monthly AFP (ICER: 27,600 pounds per QALY gained). The addition of ultrasound to this strategy increased the ICER to 60,100 pounds per QALY gained. Surveillance appears most cost-effective in individuals with hepatitis B-related cirrhosis, potentially due to younger age at diagnosis of cirrhosis. Our results suggest that, in a UK NHS context, surveillance of individuals with cirrhosis for HCC should be considered effective and cost-effective. The economic efficiency of different surveillance strategies is predicted to vary markedly according to cirrhosis aetiology.