Effect of polypeptide hormones (insulin, thyrotropin, gonadotropin, adrenocorticotropin) on RNA synthesis in Tetrahymena, as assessed from incorporation of 3H-uridine.
Incorporation of 3H-uridine by RNA in Tetrahymena was differently influenced by insulin, glucagon, follicle-stimulating hormone (FSH), thyrotropic hormone (TSH), adrenocorticotropic hormone (ACTH) and chorion-gonadotropic hormone (PMSG). TSH caused it to increase considerably and durably after an initial depression, while glucagon caused it to rise over the control throughout. Insulin, and especially PMSG, depressed the incorporation of label considerably, the latter to 3-6% of the control value by 120 min. ACTH and FSH accounted for an initial depression of RNA synthesis which, however, returned to normal 30 min after treatment. Remarkably, while the chemically similar hormones acted differently, insulin and glucagon showed the same trend of positive and negative influence, respectively.


Impact of coexistent Alzheimer pathology on the natural history of Parkinson's disease.
To assess the impact of coexisting Alzheimer (AD) pathology on the natural history of Parkinson's disease (PD). AD changes are frequently present in brains of demented PD patients. Assessing the relative contribution of AD pathology to the natural history of PD is difficult and the impact of both AD and cortical Lewy body (LB) pathologies on cognitive dysfunction is still under discussion. From clinical experience, dementia in PD patients, mainly related to AD pathology, is associated with a poor outcome, but the impact of AD pathology on the natural history of PD has not been studied systematically. In 200 consecutive autopsy cases of PD (sex (m/f) ratio 1:1.1), age at death 58-98 (mean 77.0 +/- 9.5) years, from a specialized Austrian brain bank, retrospectively assessed major initial clinical symptoms (tremor, akinesia), moderate/severe dementia, and duration of illness were correlated with associated AD pathologies using CERAD, Braak and NIA-Reagan criteria. Mann-Whitney U-test, Cox-regression were used for statistical analysis. While gender had no influence on the clinical motor symptoms and outcome, tremor dominant type had a significantly better outcome than akinetic forms (p = 0.022), even after adjustment with age at onset and associated AD pathology (CERAD and Braak criteria). Patients with late onset showed significantly shorter duration of illness irrespective of dementia. Moderate to severe dementia, reported in 33% of the sample, was significantly correlated with AD pathology (all 3 criteria) that showed significantly negative correlation with survival: between CERAD 0-A vs. B and C there was a significant difference of odd ratios (p < 0.001), as was between Braak stages 0-2, 3-4.5, and 5, but not between Braak stages 3-4 and 5. The present data confirm previous studies suggesting better outcome of tremor-dominant than akinetic-rigid type of PD, significantly worse outcome in PD with late onset and dementia that is significantly correlated with coexistent neuritic Alzheimer pathology, particularly when using the CERAD and NIA-R criteria for the diagnosis of AD. Further studies are needed to elucidate the relative impact of cortical LB and AD pathologies on the natural history of PD.


A consolidated bio-processing of ethanol from cassava pulp accompanied by hydrogen production.
A biphasic fermentation approach was undertaken for the production of ethanol and hydrogen from cassava pulp. The glucose generated by co-culture of Clostridium thermocellum and Thermoanaerobacterium aotearoense was 13.65±0.45 g L(-1), which was 1.75 and 1.17-fold greater than that produced by mono-cultures of C. thermocellum and T. aotearoense, respectively. The accumulated glucose could be utilised rapidly by subsequently inoculated Saccharomyces cerevisiae. An inoculum ratio of 1:1, a thermophilic fermentation of 84 h, and a pulp concentration of 4% proved optimal for ethanol production, fermentation efficiency, and productivity. With these conditions, the ethanol level reached 8.83±0.31 g L(-1) with a fermentation efficiency of 64.95±2.71%. Hydrogen production of 4.06 mmol by the co-culture system was 1.54 and 2.09-fold greater than that produced by mono-cultures of C. thermocellum and T. aotearoense, respectively. This sequential co-culture approach provided a consolidated bio-processing means to produce ethanol and hydrogen from cassava pulp.


Biomarkers of oxidative stress are significantly elevated in Down syndrome.
There is convincing epidemiological and in vitro evidence of chronic oxidative stress in individuals with Down syndrome (DS). These individuals develop Alzheimer like changes in the brain in their 30s and 40s. The incidence of autoimmune diseases and cataracts is significantly increased, and the overall ageing process is accelerated. In vitro studies show that impaired viability of DS neurons may be amended by simple chemical antioxidants, such as vitamin E, BHT and propyl gallate, clearly indicative of oxyl radical involvement. However, because of the lack of in vivo experiments, the role of oxidative stress in DS remains controversial. We report here on the results of the chemical analyses of urine samples of 166 individuals, where DS subjects were matched by their siblings. The levels of 8-hydroxy-2'-deoxyguanosine (2.35 +/- 1.69 in DS vs. 1.35 +/- 1.04 in controls, P = 0.00011), a biomarker of oxidative damage to DNA, and malondialdehyde (0.255 +/- 0.158 in DS vs. 0.204 +/- 0.128 in controls, P = 0.033), a biomarker of lipid peroxidation, are significantly elevated in individuals with DS. Dietary influences failed to show any significant correlation with the oxidative stress biomarkers. These results provide direct evidence for increased oxidative stress in individuals with DS.


Correction of cellular phenotypes of Hutchinson-Gilford Progeria cells by RNA interference.
The great majority of cases of the Hutchinson-Gilford progeroid syndrome (HGPS) ("Progeria of Childhood'') are caused by a single nucleotide mutation (1824 C->T) in the LMNA gene which encodes lamin A and C, nuclear intermediate filaments that are important components of the nuclear lamina. The resultant mutant protein (Delta50 lamin A) is thought to act in a dominant fashion. We exploited RNA interference technology to suppress Delta50 lamin A expression, with the long range goal of intervening in the pathogenesis of the coronary artery atherosclerosis that typically leads to the death of HGPS patients. Short hairpin RNA (shRNA) constructs were designed to target the mutated pre-spliced or mature LMNA mRNAs, and were expressed in HGPS fibroblasts carrying the 1824 C->T mutations using lentiviruses. One of the shRNAs targeted to the mutated mRNA reduced the expression levels of Delta50 lamin A to 26% or lower. The reduced expression was associated with amelioration of abnormal nuclear morphology, improvement of proliferative potential, and reduction in the numbers of senescent cells. These findings provide a rationale for potential gene therapy.


Evaluating the Performance Improvement Preferences of Disability Service Managers: An Exploratory Study Using Gilbert's Behavior Engineering Model.
Front-line managers play an important role in managing the performance of staff working in services for people with intellectual disability, but little is known about the practices they prefer to use to improve staff performance and whether these align with what research has shown to be effective. This study comprised two phases. First, the present authors tested the validity and reliability of a short questionnaire designed to evaluate managers' preferences for performance improvement practices. Then, the present authors collected and analysed responses from 175 managers working in disability services in Queensland, Australia. The questionnaire demonstrated good content validity, concurrent validity and test-retest reliability. The participants believed strategies related to changing employee individual characteristics to be more effective than strategies aimed at improving environmental factors. This study provides important considerations regarding the professional development needs of front-line managers working in organizations that provide services to people with intellectual disability.


Hypoxia impairs visual acuity in snapper (Pagrus auratus).
We investigated the effect of environmental hypoxia on vision in snapper (Pagrus auratus). Juvenile snapper inhabit estuarine environments where oxygen conditions fluctuate on a seasonal basis. Optomotor experiments demonstrated that visual acuity is impaired by environmental hypoxia, but not until levels approach the critical oxygen tension (P crit) of this species (around 25% air-saturated seawater). In 100, 80, and 60% air-saturated seawater, a positive optomotor response was present at a minimum separable angle (M SA) of 1°. In 40% air-saturated seawater, vision was partially impaired with positive responses at M SAs of 2° and above. However, in 25% air-saturated seawater, visual acuity was seriously impaired, with positive responses only present at M SAs of 6° and above. Snapper were found to possess a choroid rete, facilitating the maintenance of high ocular oxygen partial pressures (PO2) during normoxia and moderate hypoxia (PO2, between 269 and 290 mmHg). However, at 40 and 25% water oxygen saturation, ocular PO2 was reduced to below 175 mmHg, which is perhaps linked to impairment of visual acuity in these conditions. The ability to preserve visual function during moderate hypoxia is beneficial for the maintenance of a visual lifestyle in the fluctuating oxygen environments of estuaries.


Pulmonary response to airway instillation of autologous blood in horses.
Exercise-induced pulmonary haemorrhage (EIPH) occurs in the majority of horses performing strenuous exercise. Associated pulmonary lesions include alveolar and airway wall fibrosis, which may enhance the severity of EIPH. Further work is required to understand the pulmonary response to blood in the equine airways. To confirm that a single instillation of autologous blood into horse airways is associated with alveolar wall fibrosis, and to determine if blood in the airways is also associated with peribronchiolar fibrosis. Paired regions of each lung were inoculated with blood or saline at 14 and 7 days, and 48, 24 and 6 h before euthanasia. Resulting lesions were described histologically and alveolar and airway wall collagen was quantified. The main lesion observed on histology was hypertrophy and hyperplasia of type II pneumocytes at 7 days after blood instillation. This lesion was no longer present at 14 days. There were no significant effects of lung region, treatment (saline or autologous blood instillation), nor significant treatment-time interactions in the amount of collagen in the interstitium or in the peribronchial regions. A single instillation of autologous blood in lung regions is not associated with pulmonary fibrosis. Pulmonary fibrosis and lung remodelling, characteristic of EIPH, are important because these lesions may enhance the severity of bleeding during exercise. A single instillation of autologous blood in the airspaces of the lung is not associated with pulmonary fibrosis. Therefore the pulmonary fibrosis described in EIPH must have other causes, such as repetitive bleeds, or the presence of blood in the pulmonary interstitium in addition to the airspaces. Prevention of pulmonary fibrosis through therapeutic intervention requires a better understanding of these mechanisms.


Euploidy rates among preimplantation genetic testing for aneuploidy cycles with oral dydrogesterone primed ovarian stimulation or GnRH antagonist protocol.
Do differences exist in euploidy rates in preimplantation genetic testing for aneuploidy (PGT-A) cycles with oral dydrogesterone primed ovarian stimulation protocol or the flexible gonadotropin-releasing hormone (GnRH) antagonist protocol? A retrospective cohort study. Patients received the oral dydrogesterone or the GnRH antagonist in the first PGT-A cycle between November 2017 and May 2019. Propensity matching was used to identify a propensity-matched antagonist group based on age, BMI and AMH with a 1:1 ratio. The primary outcome was the rate of euploid embryos. A total of 780 cycles were included, consisting of 390 cycles receiving dydrogesterone and 390 cycles receiving GnRH antagonist protocol. No significant difference was found in patient baseline and cycle characteristics in the two groups. No statistical difference was found in the number of oocytes retrieved, metaphase II oocytes, embryos biopsied and embryo testing between the two groups. As no biopsy blastocysts formed in some cycles, only 262 cycles in the study group and 263 cycles in the antagonist group received next-generation sequencing testing, respectively. Similar to our overall data, the euploid rate per embryo biopsied was not significantly different. No significant differences were found between the two groups after stratifying by age and controlling for PGT-A testing modality. Ovulation inhibition by exogenous progestins in ovarian stimulation cycles should, therefore, be considered a valid modality in freeze-all PGT-A cycles, in view of its demonstrated effectiveness and known safety enhancement.


Complete mutation scan of the human Fas ligand gene: linkage studies in Type I diabetes mellitus families.
Type I (insulin-dependent) diabetes mellitus is the result of a T-cell regulated selective destruction of pancreatic beta cells. There is evidence that the apoptosis inducing T-cell effector, Fas ligand (FasL) could be involved in the pathogenesis of Type I diabetes, probably because FasL-mediated apoptosis is important in maintaining peripheral self-tolerance and in down-regulating an immune response. We therefore evaluated the human FasL gene FASL on chromosome 1q23 as a candidate susceptibility gene for Type I diabetes. The entire FASL (promoter, exons 1-4 and 3'UTR) was scanned for polymorphisms using single strand conformational polymorphism-heteroduplex analysis and direct sequencing. We identified two novel polymorphisms, a g-C843T and a g-A475T, in a negative regulatory region of the promoter. A Danish Type I diabetes family collection of 1143 subjects comprising 257 families (420 affected and 252 unaffected offspring) was typed for the g-C843T polymorphism and for a FASL microsatellite. Haplotypes were established and data were analysed using the extended transmission disequilibrium test. We found no overall evidence for linkage in the presence of association of the FASL polymorphism to Type I diabetes and conclude that FASL does not contribute to the genetic susceptibility to Type I diabetes.


Role of high sensitivity C-reactive protein and other risk factors in intracranial and extracranial artery occlusion in patients with ischaemic stroke.
A retrospective case-control study to identify stroke-associated risk factors and quantify serum C-reactive protein in patients with ischaemic stroke, with or without intracranial and/or extracranial artery occlusion (IEAO). Patients with ischaemic stroke and internal carotid artery occlusion (ICAO), middle cerebral artery occlusion (MCAO), ICAO + MCAO, or no IEAO (control patients) were retrospectively recruited. Data regarding stroke-associated risk factors were retrieved from medical records. High sensitivity (hs)-CRP was quantified within 3 days of hospital admission. Patients with ICAO (n = 89), MCAO (n = 74) and ICAO + MCAO (n = 29) had significantly higher serum hs-CRP concentrations, and were significantly more likely to have coronary heart disease, a history of stroke, and more than three stroke-associated risk factors than control patients (n = 84). Coronary heart disease and a history of stroke are risk factors for ischaemic stroke with IEAO. Hs-CRP may be used as a marker for IEAO.


Generation of non-human primate CAR Tregs using artificial antigen-presenting cells, simian tropic lentiviral vectors, and antigen-specific restimulation.
It is technically challenging to generate large doses of regulatory T cells (Tregs) engineered to express a chimeric antigen receptor (CAR) in non-human primates (NHP). Here, we have optimized the manufacturing of CAR Tregs by stringent sorting of Tregs, stimulation by artificial antigen-presenting cells, transduction by simian tropic lentiviral vectors, and antigen-specific expansion. The result of this method is highly suppressive CAR Tregs for use in a pre-clinical, large animal model of transplant tolerance. For complete details on the use and execution of this protocol, please refer to Ellis et al. (2022).


A Comparison of In Vitro and In Vivo Asexual Embryogenesis.
In plants, embryogenesis generally occurs through the sexual process of double fertilization, which involves a haploid sperm cell fusing with a haploid egg cell to ultimately give rise to a diploid embryo. Embryogenesis can also occur asexually in the absence of fertilization, both in vitro and in vivo. Somatic or gametic cells are able to differentiate into embryos in vitro following the application of plant growth regulators or stress treatments. Asexual embryogenesis also occurs naturally in some plant species in vivo, from either ovule cells as part of a process defined as apomixis, or from somatic leaf tissue in other species. In both in vitro and in vivo asexual embryogenesis, the embryo precursor cells must attain an embryogenic fate without the act of fertilization. This review compares the processes of in vitro and in vivo asexual embryogenesis including what is known regarding the genetic and epigenetic regulation of each process, and considers how the precursor cells are able to change fate and adopt an embryogenic pathway.


Voltage dependence of cellular current and conductances in frog skin.
Knowledge of the voltage dependencies of apical and basolateral conductances is important in determining the factors that regulate transcellular transport. To gain this knowledge it is necessary to distinguish between cellular and paracellular currents and conductances. This is generally done by sequentially measuring transepithelial current/voltage (It/Vt) and conductance/voltage (gt/Vt) relationships before and after the abolition of cellular sodium transport with amiloride. Often, however, there are variable time-dependent and voltage-dependent responses to voltage perturbation both in the absence and presence of amiloride, pointing to effects on the paracellular pathway. We have here investigated these phenomena systematically and found that the difficulties were significantly lessened by the use of an intermittent technique, measuring It and gt before and after brief (less than 10 sec) exposure to amiloride at each setting of Vt. I/V relationships were characterized by these means in frog skins (Rana pipiens, Northern variety, and Rana temporaria). Cellular current, Ic, decreased with hyperpolarization (larger serosa positive clamps) of Vt. Derived Ic/Vt relationships between Vt = 0 and 175 mV (serosa positive) were slightly concave upwards. Because values of cell conductance, gc, remained finite, it was possible to demonstrate reversal of Ic. Values of the reversal potential Vr averaged 156 +/- 14 (SD, n = 18) mV. Simultaneous microelectrode measurements permitted also the calculation of apical and basolateral conductances, ga and gb. The apical conductance decreased monotonically with increasing positivity of Vt (and Va). In contrast, in the range in which the basolateral conductance could be evaluated adequately (Vt less than 125 mV), gb increased with more positive values of Vt (and Vb). That is, there was an inverse relation between gb and cellular current at the quasi-steady state, 10-30 sec after the transepithelial voltage step.


[Expression of goose interleukin-2 gene in Escherichia coli and isolation of its soluble monomer].
Recombinant expression plasmid of pET-28a (+)-goIL-2 was constructed by inserting the goose IL-2 gene without the signal peptide sequence into the prokaryotic expression vector pET-28a (+), and transformed into the bacterial competent E. coli BL21 (DE3) cells for expression. After IPTG induction, an expected protein band with molecular weight of 15.0 kD was observed on SDS-PAGE gel, recognized by monoclonal antibody against goose IL-2 in western-blotting assay. In the pET-28a (+) expression system, much of the recombinant goose IL-2 (rgoIL-2) was found in inclusion bodies with a portion of soluble protein. The monomer and multimers of soluble goose interleukin 2 proteins were observed in native electrophoresis. The rgoIL-2 proteins were purified by Ni-NTA column under a native condition. The rgoIL-2 soluble protein monomer was isolated by a quick protein isolation and purification system of AKTA FPLC and identified by native PAGE. Bioactivity analysis showed that the rgoIL-2 monomer stimulated the proliferation of goose lymphocytes in vitro. This will establish a basis for further study about the biological function and clinical application of goose IL-2.


A Bayesian assessment of polychlorinated biphenyl contamination of fish communities in the Laurentian Great Lakes.
Polychlorinated biphenyl (PCB) contamination has historically posed constraints on the recreational and commercial fishing industry in the Great Lakes. Empirical evidence suggests that PCB contamination represents a greater health risk from fish consumption than other legacy contaminants. The present study attempts a rigorous assessment of the spatio-temporal PCB trends in multiple species across the Canadian waters of the Great Lakes. We applied a Bayesian modelling framework, whereby we initially used dynamic linear models to delineate PCB levels and rates of change, while accounting for the role of fish length and lipid content as covariates. We then implemented Bayesian hierarchical modelling to evaluate the temporal PCB trends during the dreissenid pre- and post-invasion periods, as well as the variability among and within the water bodies of the Great Lakes system. Our analysis indicates that Lake Ontario is characterized by the highest PCB levels among nearly all of the fish species examined. Historically contaminated local areas, designated as Areas of Concern, and embayments receiving riverine inputs displayed higher concentrations within each of the water bodies examined. The general temporal trend across the Great Lakes was that the high PCB concentrations during the early 1970s followed a declining trajectory throughout the late 1980s/early 1990s, likely as a result of the reductions in industrial emissions and other management actions. Nonetheless, after the late 1990s/early 2000s, our analysis provided evidence of a decline in the rate at which PCB concentrations in fish were dropping, accompanied by a gradual establishment of species-specific, steady-state concentrations, around which there is considerable year-to-year variability. The overall trends indicate that reduced contaminant emissions have brought about distinct beneficial changes in fish PCB concentrations, but past historical contamination along with other external or internal stressors (e.g., invasive species, climate change) continue to modulate the current levels, thereby posing potential risks to humans through fish consumption.


Utility of endoscopic biopsy samples to quantitate human duodenal ion transport.
Duodenal mucosal bicarbonate secretion (DMBS) prevents acid-peptic damage and facilitates nutrient absorption. DMBS is diminished in patients with duodenal ulcers and is normalized after Helicobacter pylori eradication. The measurement of DMBS in human patients in vivo requires intubation with a multi-lumen balloon tube and permits limited testing with putative agonists and antagonists. Our purpose was to develop a means to investigate transport events in human duodenal biopsy samples in vitro. After validation studies in a modified mini-Ussing chamber were performed, duodenal transport events were examined in proximal endoscopic biopsy samples from normal volunteers (n = 17). Tissues were mounted in modified mini-Ussing chambers (volume 2.5 ml, surface area 3.8 mm2). Short circuit current (Isc), potential difference (PD), and bicarbonate secretion were determined under basal conditions and after stimulation with graded doses of prostaglandin E2 (PGE2)(10(-8) to 10(-4) mol/L) and dibutyryl cAMP (db-cAMP)(10(-4) to 10(-2) mol/L). Duodenal tissues remained viable for at least 2 hours and exhibited stable basal HCO3(-) secretion and electrical parameters. Stimulation with PGE2 and db-cAMP resulted in dose-related increases in both Isc and HCO3(-) secretion (P < .05) that were abolished by ouabain and anoxia. It is concluded (1) that human duodenal bulb biopsy samples maintain their inherent transport function in mini-Ussing chambers and (2) that by using this novel method it will be possible to define the transport events that modulate human duodenal secretion, in particular bicarbonate secretion, in both health and disease.


Native-mimicking in vitro microenvironment: an elusive and seductive future for tumor modeling and tissue engineering.
Human connective tissues are complex physiological microenvironments favorable for optimal survival, function, growth, proliferation, differentiation, migration, and death of tissue cells. Mimicking native tissue microenvironment using various three-dimensional (3D) tissue culture systems in vitro has been explored for decades, with great advances being achieved recently at material, design and application levels. These achievements are based on improved understandings about the functionalities of various tissue cells, the biocompatibility and biodegradability of scaffolding materials, the biologically functional factors within native tissues, and the pathophysiological conditions of native tissue microenvironments. Here we discuss these continuously evolving physical aspects of tissue microenvironment important for human disease modeling, with a focus on tumors, as well as for tissue repair and regeneration. The combined information about human tissue spaces reflects the necessities of considerations when configuring spatial microenvironments in vitro with native fidelity to culture cells and regenerate tissues that are beyond the formats of 2D and 3D cultures. It is important to associate tissue-specific cells with specific tissues and microenvironments therein for a better understanding of human biology and disease conditions and for the development of novel approaches to treat human diseases.


Extract from Clerodendron colebrookianum Walp protects rat heart against oxidative stress induced by ischemic-reperfusion injury (IRI).
Reactive oxygen species (ROS) have pathogenic effects on ischemic-reperfusion injury of heart. Hence, it is important to identify natural antioxidative agents to mitigate such effects. Recently, it has been reported that Clerodendron colebrookianum (CC) leaf extract has antioxidant and hypolipidemic effects in experimental animals. The aim of this study was to examine whether acute treatment with CC extract offers protection against ischemic-reperfusion injury (IRI) and IRI-induced changes in endogenous antioxidant enzyme activities of rat heart. Isolated rat hearts were perfused using the Langendorff's technique, and 20 min of global ischemia was followed by 40 min of reperfusion. Lipid peroxidation after the ischemic-reperfusion episode was significantly reduced in the CC extract-treated heart compared to the control group and suppressed the leakage of lactate dehydrogenase (LDH) during reperfusion. Moreover, CC extract diminished the depletion of myocardial antioxidant enzymes (SOD, Catalase, GSH and GPx) after ischemia-reperfusion. Furthermore, IRI-induced cellular damage was significantly less in CC extract treated myocytes. These results indicate that CC leaf extract protects against oxidative stress and cellular injury associated with ischemic-reperfusion injury of rat heart and suggests that the protective effects of CC extract depend on its antioxidant properties.


Adjuvant portal liver infusion with 5-fluorouracil and mitomycin in colorectal cancer. SAKK.
Between 1981 and 1987, 533 patients from 9 institutions were entered in a randomized trial to assess the value of adjuvant perioperative portal infusion with Fluorouracil and Mitomycin as compared to radical surgery alone. Analysis of 469 evaluable patients at a median follow-up of 5.8 years revealed 110 recurrences in the control and 94 recurrences in the infusion group. Estimated 5-year disease-free survival was 52% and 61% respectively (hazard ratio: 1:0.75; 95% confidence interval 0.57-0.99; p = 0.046). Overall survival was 59% in the control and 69% in the infusion group (p = 0.048). Adjuvant portal infusion did not influence the occurrence of liver metastases but reduced the overall recurrence rate.


Occludin is required for apoptosis when claudin-claudin interactions are disrupted.
Disruption of tight junctions is often seen during pathogen infection, inflammation, and tumor progression. Mislocalization of the tight junction proteins occludin and claudin in mammary epithelial monolayers leads to apoptosis through the extrinsic pathway. To further investigate the mechanism of this response, a normal mammary epithelial cell line (EpH4) as well as primary mammary epithelial cells were treated with a claudin-disrupting mimic peptide, DFYNP (aspartic acid-phenylalanine-tyrosine-asparagine-proline). Using fluorescent indicators, we found that caspase-3 activation, resulting from treatment with DFYNP, was restricted to EpH4 and primary mammary epithelial cells with mislocalized claudin-4. Mislocalized claudin-4 and occludin were colocalized in non-junctional puncta, and both molecules were found in the death-inducing signaling complex (DISC) where they colocalized with Fas, fas-associated protein with death domain (FADD), active caspase-8 and caspase-3 at distinct apical domains. Importantly, caspase-3 activation was totally repressed in primary mammary epithelial cells from occludin null mice. Thus, the apoptotic response appears to be initiated by the movement of occludin to the DISC suggesting that this molecule has signaling properties that initiate cell death when its tight junction location is disrupted.


Additive fabrication and experimental validation of a lightweight thermoelectric generator.
We develop a thermoelectric generator based on catalytic combustion and operating in the low power range (up to 10 W). Considering the target of small-scale thermoelectric generators, the additive technique was chosen as an enabling technology to customize the different parts of the presented device. The generator consists of a hexagonal shaped combustion chamber coupled to commercial thermoelectric modules, water-cooled at the cold side. Thanks to the components design, heat transfer across each part of the system is properly driven enhancing the thermal management of the system. Moreover, in order to improve the overall efficiency, exhausts outlet is designed to promote heat recovery. The generator is characterized achieving an electrical power output close to 9 W in continuous regime, with an overall efficiency of 3.55%. The compact size, the light weight, the simple design and the reliability in continuous operating conditions are all promising features of the device described. Furthermore, the materials chosen for the device can suggest a way to fabricate cheaper heat exchangers, actually one of the main costs of the device development.


Studies on the location and release of cholecystokinin and vasoactive intestinal peptide in rat and cat spinal cord.
By radioimmunoassay vasoactive intestinal peptide (VIP) and cholecystokinin (CCK) are found in the cat lumbar spinal ganglion and spinal cord with levels in dorsal greater than ventral horn. Unilateral rhizotomy, but not cervical hemisection produced a significant but incomplete depletion of CCK and VIP immunoreactivity in dorsal, but not ventral horn. Intrathecal capsaicin (0.5 mg) had no effect on the levels of spinal VIP or CCK. Intrathecal colchicine (0.5 mg)produced a significant increase in the levels of VIP in the dorsal and ventral horn but had no effect on the levels of CCK. The present experiments, using a preparation which permits in situ superfusion of the spinal cord, demonstrated in the chloralose-urethanized cat and rat the presence of measurable levels of VIP and CCK. In rats, the addition of potassium (40 mM in excess) resulted in a 138% and 46% increase in the levels of CCK and VIP, respectively above resting levels (3.7 +/- 1.2 fmol/ml/10 min and 1.7 +/- 0.5 fmol/ml/10 min, respectively). The deletion of calcium and substitution of cobalt (2 mM) resulted in a significant reduction in the potassium-evoked release. Intrathecal picrotoxin doubled the levels of CCK, but had no effect on the levels of VIP in the spinal superfusates. Capsaicin (3 X 10(-4) M) had no effect on the levels of either peptide in rat spinal superfusate. In cats, bilateral electrical stimulation of the sciatic nerve at high, but not low intensity, resulted in a 218% and 132% increase above prestimulation baseline in the levels of CCK and VIP, respectively. Separation of immunoreactivity on a Sephadex G-50 superfine column of the spinal superfusates and the extracted material from cat spinal cord, revealed that the immunoreactive CCK species in tissue co-migrated with the 8 and 33 amino acid peptide fragments. In the release samples, however, all the radioimmunoassayable activity migrated with the peak corresponding with CCK. No other peaks were detected. Column separation of spinal cord and the superfusate obtained during basal and evoked release, revealed that all activity in both the tissue and perfusate samples, travelled in a single peak which co-migrated with authentic VIP.


Synergistic anti-tumor efficacy of a hollow mesoporous silica-based cancer vaccine and an immune checkpoint inhibitor at the local site.
Immune checkpoint inhibitors elicit durable tumor regression in multiple types of tumor, but may induce potential side effects with low response rates in many tumors. Herein, to increase the therapeutic efficacy of immune checkpoint inhibitors, a hollow mesoporous silica (HMS) nanosphere-based cancer vaccine was combined with an immune checkpoint inhibitor, anti-programmed death-ligand 1 (anti-PD-L1) antibody. The HMS nanospheres function as adjuvants that promote dendritic cell activation and antigen cross-presentation. Mice immunized with the HMS-based cancer vaccine show suppressed tumor growth with increased tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-2 (IL-2) levels in their spleens compared with those without HMS-based cancer vaccine. Moreover, the HMS-based cancer vaccine synergistically acts with the anti-PD-L1 antibody on the tumor. The combination of an HMS-based cancer vaccine and an antibody markedly decreases the required dose of the immune checkpoint inhibitor. Mice locally administered with the HMS-based cancer vaccine and 1/8 dose of a standard anti-PD-L1 antibody (25 µg/mouse) show comparable anti-tumor effect and significantly increased CD4+ and CD8+ T cell populations, compared with those systemically immunized with the standard anti-PD-L1 antibody done at 200 µg/mouse. Our work presents a promising cancer treatment strategy of combining an immune checkpoint inhibitor with an HMS-based cancer vaccine. STATEMENT OF SIGNIFICANCE: The clinical benefits of checkpoint blockade therapy rekindle the hope of cancer immunotherapy. However, objective response rates in checkpoint blockade therapy remain at about 10-40% owing to multiple immunosuppressive factors. To solve these problems, herein, a hollow mesoporous silica (HMS) nanosphere-based cancer vaccine was combined with an immune checkpoint inhibitor, anti-PD-L1 antibody. The HMS-based cancer vaccine synergistically acts with the anti-PD-L1 antibody on the tumor. Mice locally administered with the HMS-based cancer vaccine and 1/8 dose of a standard anti-PD-L1 antibody (25 µg/mouse) show comparable anti-tumor effect and significantly increased CD4+ and CD8+ T cell populations, compared with those systemically immunized with the standard anti-PD-L1 antibody done at 200 µg/mouse. Our work presents a promising cancer treatment strategy of combining an immune checkpoint inhibitor with an HMS-based cancer vaccine.


Epicatechin gallate, a component of green tea, reduces halotolerance in Staphylococcus aureus.
Staphylococcus aureus grows in the presence of high-salt concentrations. The green tea polyphenolic compound epicatechin gallate (ECg) extended the staphylococcal lag phase to values greater than 6 h in the presence of NaCl, KCl and LiCl; this effect was not observed with epicatechin. Osmoprotectants glycine betaine and l-proline were unable to relieve the effect of ECg on halotolerance. The capacity of ECg to suppress staphylococcal growth in the presence of salt suggests that this molecule could be used to aid the preservation of salt-containing foods.


Substrate regulation of calcium binding in Ca2+-ATPase molecules of the sarcoplasmic reticulum. II. Effect of CTP, GTP, ITP, and UTP.
To examine the effect of CTP, GTP, ITP, and UTP on calcium binding of Ca2+-ATPase molecules of the sarcoplasmic reticulum, the calcium dependence of the Ca2+-activated hydrolysis activities of these NTPs of the enzyme molecules was examined by comparison with that of calcium binding of the molecules in the absence of the NTPs at pH 7.40. In the sarcoplasmic reticulum membrane, CTP, GTP, and ITP did not affect the noncooperative (Hill value (n(H)) of approximately 1, apparent calcium affinity (K(0.5)) of 2-6 microm)) and cooperative (n(H) approximately 2, K(0.5) approximately 0.2 microm) calcium binding of the molecules, whereas UTP caused the molecules to highly cooperatively (n(H) approximately 4) bind calcium ions with a lowered K(0.5) of approximately 0.04 microm. When the enzyme molecules were solubilized with detergent, all of these NTPs reversibly degraded the calcium affinity of the molecule (from K(0.5) = 3-5 to >40 microm), although the effect of the NTPs on the negatively cooperative manner (n(H) approximately 0.5) of calcium binding was not experimentally obtained. Taking into account the first part of this study (Nakamura, J., Tajima, G., Sato, C., Furukohri, T., and Konishi, K. (2002) J. Biol. Chem. 277, 24180-24190) showing the improving effect of ATP on calcium binding of the membranous and solubilized molecules, the results show that ATP is the only intrinsic substrate for the enzyme molecule. This NTP regulation is discussed in terms of the oligomeric structure of the molecules.


Evolutionary shift toward protein-based architecture in trypanosomal mitochondrial ribosomes.
Ribosomal RNA (rRNA) plays key functional and architectural roles in ribosomes. Using electron microscopy, we determined the atomic structure of a highly divergent ribosome found in mitochondria of Trypanosoma brucei, a unicellular parasite that causes sleeping sickness in humans. The trypanosomal mitoribosome features the smallest rRNAs and contains more proteins than all known ribosomes. The structure shows how the proteins have taken over the role of architectural scaffold from the rRNA: They form an autonomous outer shell that surrounds the entire particle and stabilizes and positions the functionally important regions of the rRNA. Our results also reveal the "minimal" set of conserved rRNA and protein components shared by all ribosomes that help us define the most essential functional elements.


Efficiency enhancement of dye-sensitized solar cells by interface modification between photoanode and electrolyte.
A nanoporous TiO2 layer on a transparent electrode was modified with an aqueous lithium hydroxide solution by a simple dipping process, and effects of the modified electrode on the photovoltaic properties of dye-sensitized solar cells were investigated. The modification led to a considerable improvement of 23.6% in power conversion efficiency due to an increase in both open circuit voltage and fill factor. From the measurements of the electronic band structure, electrochemical impedance spectra and incident photon-to-current conversion efficiency, it was revealed that the modification by LiOH formed a surface dipole on the TiO2 electrode, leading to a negative shift of conduction band edge of TiO2.


Galactic cosmic ray origin of Li, Be and B in stars.
A study of the implications of the hypothesis that high energy processes involving cosmic rays acting on the interstellar medium are the sources of the elements Li, Be and B present in stellar atmospheres and in the solar system.


What Are the Live Birth and Multiple Pregnancy Rates When 1 Versus 2 Low-Quality Blastocysts Are Transferred in a Cryopreserved Cycle? a Retrospective Cohort Study, Stratified for Age, Embryo Quality, and Oocyte Donor Cycles.
Outcomes among women who transferred only Gardner's grade BB or lower quality frozen embryos transferred (FET) are not well known. Our objective is to study whether transferring 2 versus 1 frozen low-quality blastocysts will increase the live birth rate (LBR) and the multiple pregnancy rate (MPR). This is a retrospective cohort study including 1104 FET cycles. Only day 5-6 blastocysts of grade BB or lower quality were included. Clinical pregnancy rate (CPR), MPR, and LBR per cycle were compared between single embryo transfer (SET) (n = 969) and double embryo transfer (DET) (n = 135). CPR and MPR were compared between SET and DET in grade BB, BC, CB, and CC individually. Among SET, BB blastocysts had higher CPR 34% (P = 0.0001) and a sub-significant increase in LBR 19% (P = 0.059) in comparison to other grade SET. Among all BB, MPR was significantly higher when transferring two versus one (5.9 vs. 1.9, P = 0.009). If age at egg collection ≥ 40 years (n = 97), no difference was found in CPR (11.1 vs. 11.7, P = 0.9), MPR (0 vs. 0), and LBR (6.3 vs. 0,P = 0.13) when SET or DET was performed. If age was < 40 years (n = 818), the MPR was significantly higher in DET than SET (6.7 vs. 1.63, P = 0.004). In egg donor cycles (n = 189), there was no difference in CPR, MPR, and LBR between SET and DET. Single embryo transfer should be offered even in women ≥ 40 years of age or transferring lower quality embryos since transferring more did not increase outcomes in this group, and SET is likely the safest path.


Stable carbon isotope ratios in Asian elephant collagen: implications for dietary studies.
Stable carbon isotope ratios in bone collagen have been used in a variety of dietary studies in modern and fossil animals, including humans. Inherent in the stable isotope technique is the assumption that the isotopic signature is a reflection of the diet and is persistent in collagen because this is a relatively inert protein. Carbon isotope analyses of bones from a southern Indian population of Asian elephant (Elephas maximus), a long-lived mammal that alternates seasonally between a predominantly C3 (browse) and C4 (grass) plant diet, showed two patterns that have important implications for dietary interpretation based on isotopic studies. Relative to the quantity of the two plant types consumed on average, the δ13C signal in collagen indicated that more carbon was incorporated from C3 plants, possibly due to their higher protein contribution. There was a much greater variance in δ13C values of collagen in sub-adult (range -10.5‰ to-22.7‰, variance=14.51) compared to adult animals (range -16.0‰ to -20.3‰, variance=1.85) pointing to high collagen turnover rates and non-persistent isotopic signatures in younger, growing animals. It thus seems important to correct for any significant relative differences in nutritive value of food types and also consider the age of an animal before drawing definite conclusions about its diet from isotope ratios.


Development of a high-sensitivity immunoassay for amyloid-beta 1-42 using a silicon microarray platform.
In this work, we present a highly sensitive immunoassay for the detection of the Alzheimer's disease (AD) biomarker amyloid-beta 1-42 (Aβ42) based on a label/label-free microarray platform that utilises silicon/silicon oxide (Si/SiO2) substrates. Due to constructive interference, Si/SiO2 layered slides allow enhancement of the fluorescence intensity on the surface with significant improvements in sensitivity of detection. The same substrate allows the label-free multiplexed detection of targets using the Interferometric Reflectance Imaging Sensor (IRIS), a platform amenable to high-throughput detection of mass changes on microarray substrates. Silicon chips are coated with copoly(DMA-NAS-MAPS), a ter-copolymer made from dimethylacrylamide (DMA), 3-(trimethoxysilyl)propyl methacrylate (MAPS) and N-Acryloyloxy succinimide (NAS). Aβ42 aggregation was studied by circular dichroism (CD), and an optimal antibody pair was selected based on specificity of recognition, binding yield and spot morphology of the capture antibody on the coated silicon surface as analysed by IRIS. Finally, incubation conditions were optimised, and an unprecedented Aβ42 detection sensitivity of 73pg/mL was achieved using an artificial cerebrospinal fluid (CSF) sample. Because of their multiplexing capability, low volume sample consumption and efficient sample-to-result time for population-wide screening, microarrays are ideal tools for the identification of individuals with preclinical AD who are still cognitively healthy. The high sensitivity of this assay format, potentially coupled to a pre-concentration step or signal-enhancing modifications, could lead to a non-invasive, inexpensive diagnostic tool for population-wide screening of AD biomarkers in biological fluids other than CSF, such as serum or plasma.


Ionic liquid coated magnetic nanoparticles for the gas chromatography/mass spectrometric determination of polycyclic aromatic hydrocarbons in waters.
In this paper, ionic liquid coated magnetic nanoparticles (IL-MNPs) have been prepared by covalent immobilization. The as-synthesized MNPs have been successfully used as sorbent for the extraction of polycyclic aromatic hydrocarbons (PAHs) from water samples, the analytes being finally determined by gas chromatography/mass spectrometry. The influence of several experimental variables (including the ionic strength, amount of MNPs, sample volume, agitation time and desorption solvent) has been considered in depth in the optimization process. The developed method, which has been analytically characterized under its optimal operation conditions, allows the detection of the analytes in the samples with method detection limits in the range from 0.04μgL(-1) (fluoranthene) to 1.11μgL(-1) (indeno(1,2,3-cd)pyrene). The repeatability of the method, expressed as relative standard deviation (RSD, n=7), varies between 4.0% (benzo[b]fluoranthene) and 8.9% (acenaphthene), while the enrichment factors are in the range from 49 (naphthalene) to 158 (fluoranthene). The proposed procedure has been applied for the determination of thirteen PAHs in water samples (tap, river, well and reservoir ones) with recoveries in the range from 75 to 102%.


Bulked Oligo-FISH for Chromosome Painting and Chromosome Barcoding.
Recently developed bulked oligo-FISH is a highly versatile method, which is applicable in any plant species with an assembled genome sequence. This technique allows in situ identification of individual chromosomes, large chromosomal rearrangements, comparative karyotype analysis, or even the reconstruction of the three-dimensional organization of the genome. The method is based on the identification of thousands of short oligonucleotides, unique to specific genome regions, which are synthesized in parallel, fluorescently labeled and used as probes for FISH. In this chapter, we propose a detailed protocol for amplification and labeling of single-stranded oligo-based painting probes from so-called MYtags immortal libraries, the preparation of mitotic metaphase and meiotic pachytene chromosome spreads, and a protocol for the fluorescence in situ hybridization procedure using the synthetic oligo probes. The proposed protocols are demonstrated for banana (Musa spp.).


Matrix metalloproteinase activation by free neutrophil elastase contributes to bronchiectasis progression in early cystic fibrosis.
Neutrophil elastase is the most significant predictor of bronchiectasis in early-life cystic fibrosis; however, the causal link between neutrophil elastase and airway damage is not well understood. Matrix metalloproteinases (MMPs) play a crucial role in extracellular matrix modelling and are activated by neutrophil elastase. The aim of this study was to assess if MMP activation positively correlates with neutrophil elastase activity, disease severity and bronchiectasis in young children with cystic fibrosis.Total MMP-1, MMP-2, MMP-7, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-2 and TIMP-1 levels were measured in bronchoalveolar lavage fluid collected from young children with cystic fibrosis during annual clinical assessment. Active/pro-enzyme ratio of MMP-9 was determined by gelatin zymography. Annual chest computed tomography imaging was scored for bronchiectasis.A higher MMP-9/TIMP-1 ratio was associated with free neutrophil elastase activity. In contrast, MMP-2/TIMP-2 ratio decreased and MMP-1 and MMP-7 were not detected in the majority of samples. Ratio of active/pro-enzyme MMP-9 was also higher in the presence of free neutrophil elastase activity, but not infection. Across the study cohort, both MMP-9/TIMP-1 and active MMP-9 were associated with progression of bronchiectasis.Both MMP-9/TIMP-1 and active MMP-9 increased with free neutrophil elastase and were associated with bronchiectasis, further demonstrating that free neutrophil elastase activity should be considered an important precursor to cystic fibrosis structural disease.


Selective inhibition of the chymotrypsin-like activity of the 20S proteasome by 5-methoxy-1-indanone dipeptide benzamides.
Potent inhibitors of the 20S proteasome that contain a novel indanone head group coupled to di and tripeptides are described. These compounds are the first proteasome inhibitors have demonstrated high selectivity for the chymotrypsin-like activity of the 20S proteasome.


β-arrestin2 mediates the hippocampal dopaminergic system in autistic mouse through the ERK signaling pathway.
Autism is a complex neurodevelopmental disease that may be caused by genetic and environmental factors, that are incompletely understood. Overactivation of dopaminergic receptors can lead to autistic-like behavior. β-arrestin2 (Arrb2) is a scaffolding protein of the arrestin family, which function as cytosolic multifunctional adapter proteins that activate cell signal transduction and mediate the signal termination and endocytosis of G-protein-coupled receptors (GPCRs) complexes. In this study, we established an Arrb2 knockout (Arrb2-/-) mouse to explore the biological function of Arrb2 in autistic-like behavior caused by abnormality in the dopaminergic system. We found that Arrb2-/- mice did not exhibit the autistic-like behavior normally induced by SKF38393, an agonist of the dopamine receptor 1 (D1R). Compared with wild-type (WT) untreated mice, the SKF38393-treated WT mice and Arrb2-/- mice, with or without SKF38393 treatment, showed abnormalities on electroencephalography (EEG) and increased stimulation of the phosphorylated form of extracellular signal-regulated kinase (p-ERK) via the PKA/Rap1/B-Raf/MEK pathway. These results demonstrated that Arrb2 regulated the dopaminergic system through the ERK signaling pathway in the occurrence and development of autism, and that targeted deletion of Arrb2 impeded the development of autistic-like behavior.


Quantitative model of small molecules uptake after in vitro cell electropermeabilization.
Electropermeabilization of the cell membrane is a phenomenon caused by exposure of the cell to electric pulses. Permeabilization depends on pulse duration, pulse amplitude, the number of pulses delivered, and also on other experimental conditions. With these parameters properly chosen, the process of permeabilization is reversible and cells return to their normal physiological state. This article describes the development of a model of diffusion-driven transmembrane transport of small molecules caused by electropermeabilization. The process of permeabilization is divided into a short permeabilizing phase that takes place during the pulse, and a longer resealing phase that begins after the end of the pulse. Because both phases of permeabilization are important for uptake of molecules into cells, most of the effort is focused on the optimization of parameters that influence the flow between intracellular and extracellular space. The model describes well the transmembrane transport caused by electropermeabilization, allowing to study the uptake of molecules as a function of elapsed time, voltage and pulse duration. In addition, our results show that the shapes of the curves of cell permeabilization and survival as functions of pulse amplitude can to a large extent be explained by cell size distribution.


Exploiting the hallmarks of cancer: the future conquest of breast cancer.
What separates a malignant from a normal cell? This question has occupied scientists for decades. Although a simple answer remains elusive, several hallmarks of malignancy have been identified. These critical features include uncontrolled proliferation, insensitivity to negative growth regulation, evasion of apoptosis, lack of senescence, invasion and metastasis, angiogenesis and genomic elasticity. Existing therapies predominantly target proliferation either with cytotoxic agents, ionising radiation or more targeted attacks on growth factor signalling pathways. Our most successful therapies to date inhibit proliferation via the oestrogen receptor (ER) and HER2 pathways. Further improvements in therapy must attack the other hallmarks of malignancy and will undoubtedly be accompanied by a better means of individual patient selection for such therapies. Indeed, each of these hallmarks presents a therapeutic opportunity. To believe otherwise would be to assume that a feature is both biologically crucial, yet therapeutically unimportant, an unlikely paradox. Here, we suggest the hallmarks of malignancy as a conceptual framework for understanding novel breast cancer therapies.


Studying the aging of Laponite suspensions using extensional rheology.
The effect of aging on the break-up dynamics of Laponite suspensions was studied in an extensional geometry. It was found that samples of increased age undergo stronger necking at the midpoint. The thinning of samples, driven purely by motion of the plates, was compared with standard shear rheology to understand how the dynamics are related to the sample properties. The Laponite suspensions exhibit a growing stress overshoot with monotonically decreasing yield strain as they age. However, it is shown that the thinning curves in extension are only a good indicator of the sample's static yield stress, being insensitive to its yield strain. These measurements suggest that following an initial linear visco-elastic regime, samples accumulate significant plastic deformations prior to the complete yielding of the sample. The implications of this for the importance of assessing changes to the ductile-brittle nature of samples are also discussed.


Clinical, morphologic, and clinicopathologic findings in Beagles treated for two years with melengestrol acetate.
Melengestrol acetate (MGA) was administered orally to groups of 10 female and 3 male Beagles at doses of 0, 1, 2, or 8 microgram/kg of body weight/day for 2 years. Treatment was continuous at the same dose rate in the first 3 groups, but in the 4th group, the dose for bitches was reduced to 4 microgram of MGA/kg/day during the 2nd year. Matings were made within MGA-dosage groups and among 10 additional bitches treated at 1 microgram of MGA/kg/day and the dogs treated at 8 microgram/kg/day. Doses of 8 and 4 microgram of MGS/kg caused progestational effects on the uterus resulting in expected histopathologic changes, dystocia, and pyometritis. Leukocytosis, normocytic, hypochromic anemia, and increased alkaline phosphatase values were the results of systemic and uterine effects consistent with the indirect and expected pharmacologic action of progestational agents in the bitch. Doses of 1 and 2 microgram of MGA/kg in dogs and bitches and 8 microgram/kg in dogs produced no significant differences in clinical observations, hematologic findings, blood chemical analysis, urinalysis, organ weights, or gross and microscopic observations at necropsy.


Statistical optimization of medium components for production of extracellular chitinase by Basidiobolus ranarum: a novel biocontrol agent against plant pathogenic fungi.
The influence of concentration of medium components such as colloidal chitin, lactose, malt extract, yeast extract, and peptone on the chitinase production from Basidiobolous ranarum at the flask level were studied by using statistical tool Central Composite Design (CCD) and analysed by Response Surface Methodology (RSM). The results revealed that colloidal chitin, malt extract and peptone had significant effect (P < 0.01) on the chitinase production at their individual levels. The polynomial equation of the model developed incorporates 3 linear, 3 quadratic and 5 interactive terms. Maximum chitinase production of 3.47 U ml(-1) was achieved with 1.5% colloidal chitin, 0.125% lactose, 0.025% malt extract and 0.075% peptone. After optimization, chitinase activity was increased by 7.71 fold. A second order polynomial equation was found to be useful for the development of efficient bioprocess for chitinase production. To screen the biotechnological potential of this enzyme, degradation of fungal mycelia by ammonium sulphate precipitate of the same was studied for several pathogenic fungi-in vitro which showed promising results particularly against Rhizoctonia solani and Fusarium solani. This study provides the first evidence showing the effectiveness of RSM for the development of a robust statistical model for the chitinase production by Basidiobolus and for its application in the biocontrol of phytopathogenic fungi.


Transcriptome Analysis Indicates Immune Responses against Vibrio harveyi in Chinese Tongue Sole (Cynoglossus semilaevis).
Pathogenic infection of fishes is an important constraining factor affecting marine aquaculture. Insufficient understanding of the molecular mechanisms has affected the diagnosis and corresponding treatment. Here, we reported the dynamic changes of gene expression patterns in the Chinese tongue sole kidney at 16 h, 48 h, 72 h and 96 h after Vibrio harveyi infection. In total, 366, 214, 115 and 238 differentially expressed genes were obtained from the 16 h-vs. -C, 48 h-vs. -C, 72 h-vs. -C and 96 h-vs. -C group comparisons, respectively. KEGG enrichment analysis revealed rapid up-regulation of several immune-related pathways, including IL-17, TNF and TLR signaling pathway. More importantly, time-series analyses of transcriptome showed that immune genes were specifically up-regulated in a short period of time and then decreased. The expression levels of chemokines increased after infection and reached a peak at 16 h. Specifically, Jak-STAT signaling pathway played a crucial role in the regulation during Vibrio harveyi infection. In the later stages of infection, genes in the neuroendocrine pathway, such as glucocorticoid-related genes, were activated in the kidney, indicating a close connection between the immune system and neuroendocrine system. Our dynamic transcriptome analyses provided profound insight into the gene expression profile and investigation of immunogenetic mechanisms of Chinese tongue sole.


Widely spread butyrylcholinesterase can hydrolyze acetylcholine in the normal and Alzheimer brain.
Butyrylcholinesterase (BChE), also known as the "pseudo" or "non-neuronal" cholinesterase, is traditionally thought to have a restricted CNS distribution and to play little, if any, role in cholinergic transmission. To reanalyze the role of BChE in the human brain with more sensitive methodology. Three brains were examined with acetylcholinesterase and BChE histochemistry. The sections were examined with bright- and dark-field microscopy. The histochemical parameters used in the present experiments showed that BChE activity was present in all hippocampal and temporal neocortical areas known to receive cholinergic input. At all of these locations, the BChE enzyme could hydrolyze the acetylcholine surrogate acetylthiocholine. A substantial portion of the hippocampal and neocortical BChE appeared to be located within neuroglia and their processes. Butyrylcholinesterase may have a greater role in cholinergic transmission than previously surmised, making BChE inhibition an important therapeutic goal in Alzheimer's disease. The results also suggest that the role of neuroglia in cholinergic transmission may be analogous to their well known role in glutamatergic transmission.


Decrease of the spontaneous non-quantal release of acetylcholine from the phrenic nerve in botulinum-poisoned rat diaphragm.
Botulinum type A toxin (BoTx) has been found to diminish by 40% the spontaneous release of acetylcholine (ACh) from normal (acutely denervated) rat diaphragms incubated in the presence of 5 mM K+, while the release of ACh from chronically (4 days) denervated diaphragms was not affected during 2 h incubations. The toxin has been found to rapidly remove (within 10 min) the local depolarization of about 8 mV which developed in the end-plate zones of the diaphragms after the inhibition of cholinesterases; after the administration of BoTx, tubocurarine lost its ability to increase the resting membrane potential (H-response, Katz and Miledi 1977) in the end-plate area of anticholinesterase-treated muscles. It is concluded that BoTx inhibits the non-quantal release of ACh from the motor nerve fibres and that it probably acts directly on the nerve terminal surface membrane (without internalization). The H-response in the rat diaphragm reflects the non-quantal release of ACh from the nerve terminals and not from the muscle fibres.


Efficacy and safety of sirolimus in patients with systemic lupus erythematosus: A systematic review and meta-analysis.
Emerging evidence suggested a potential therapeutic role of targeting mTOR in the treatment of SLE. But most studies were observational studies with limited sample size or case reports. To evaluate the efficacy and safety of sirolimus in treatment of SLE by systematic review and meta-analysis. Systematic searches of Medline/PubMed, EMBASE, the Cochrane library and Scopus were performed. Original case reports, case series, observational studies and clinical trials reporting the efficacy or safety data on SLE patients treated with sirolimus were included. A random-effects meta-analysis was performed to calculate the pooled efficacy, when possible. A total of 9 studies comprising 145 patients were identified. The exposure of sirolimus was 245.8 patient-years, with 1-3 mg/day adopted in majority studies. In 111 clinical active patients, the pooled decrease of SLEDAI, BILAG and prednisone dosage was 4.85 (95% CI 3.44-6.25), 1.98 (95% CI 0.23-3.74) and 13.17 mg/day (95% CI 0.71-25.63) respectively. 23 patients initiating sirolimus for active SLE yielded remission in 17 (73.9%) patients. In 22 quiescent lupus nephritis patients, 21 (95.5%) patients sustained remission. Hematological, mucocutaneous abnormalities and dyslipidemia were the most common adverse events. Early cessation due to side effects was reported in 9.28% (13/140) patients, most of the side effects were mild and recovered quickly after cessation. Summary of the available datasets indicated sirolimus was promising and well-tolerated in the treatment of SLE. Further randomized controlled trials evaluating the potential benefits and risk of sirolimus in SLE are warranted.


Chemoselective intramolecular alkylation of the Blaise reaction intermediates: tandem one-pot synthesis of exo-cyclic enaminoesters and their applications toward the synthesis of N-heterocyclic compounds.
The intramolecular alkylative reactivity and N/C selectivity of the various Blaise reaction intermediates, which are formed from the reaction of the Reformatsky reagents with ω-chloroalkyl nitriles, did not reach the synthetic potential as an entry to exo-cyclic enaminoesters. To circumvent this issue, various additives were investigated, among which the addition of NaHMDS dramatically enhanced the reactivity and N/C selectivity. This modification provided a highly efficient route for the synthesis of various N-fused heterocyclic compounds, as it requires only two steps from nitriles.


Early postoperative use of unfractionated heparin or enoxaparin is associated with increased surgical re-exploration for bleeding.
A variety of indications (eg, prosthetic heart valves, atrial fibrillation, etc.) exist for the use of unfractionated heparin (UFH) and enoxaparin (ENOX) in the early postoperative period following open-heart surgery. However, the overall postoperative risk for hemorrhage from the use of UFH and ENOX are not known. From 1998 to 2001, 2,977 consecutive open-heart or valve surgery patients were retrospectively evaluated. Postoperatively, 2,037 received no UFH or ENOX, 579 received intravenous UFH, and 361 received ENOX. Baseline characteristics were collected, patients who required surgical re-exploration for postoperative bleeding and time between surgery and re-exploration were followed-up. Average patient ages were 64 +/- 13, 65 +/- 12, and 68 +/- 10 years receiving none, UFH (p < 0.01 vs none), and ENOX (p < 0.01 vs none; p < 0.01 vs UFH), respectively. Rates of surgical re-exploration were 2.7% for none, 7.8% for UFH, and 8.9% for ENOX (vs none, adjusted hazard ratio = 2.8; p < 0.001 for UFH; hazard ratio = 3.3; p < 0.001 for ENOX). Males were also at higher risk for re-exploration (hazard ratio = 1.4; p = 0.07). For those requiring re-exploration, the interval between surgery and first re-exploration was prolonged (> 4 days) among those receiving ENOX (37.5%, odds ratio = 36.7; p = 0.001) and UFH (20.0%, odds ratio = 14.7; p = 0.01) compared with none (1.8%). Prolonged times with ENOX had a greater proportion of prolonged times than UFH (odds ratio = 2.5; p = 0.09). Early postoperative use of ENOX and UFH is associated with a significant increase in re-exploration for postoperative bleeding, often at a significantly delayed time period after the initial surgery. This delay was especially common with ENOX suggesting the need for prospective studies.


Correlated histological and physiological observations on a case of common sensory output and motor input of the bag1 fibre and a chain fibre in a cat tenuissimus spindle.
In muscle spindles of the cat, independent control of dynamic and static components of the response of the primary sensory ending to stretch is provided by separate motor inputs to the various kinds of intrafusal muscle fibre: dynamic axons (gamma or beta) to the bag1 fibres and static axons to the bag2 (typically gamma only) and chain (gamma or beta) fibres. Nonlinear summation of separately evoked effects during combined stimulation of dynamic and static motor axons appears to be due to mutual resetting by antidromic invasion of separate encoding sites, leading to partial occlusion of the momentarily lesser response by the greater. The encoding sites are thought to be located within the primary ending's preterminal branches which from first-order level are normally segregated to the bag1 fibre and to the bag2 and chain fibres. Here we describe the analysis of a special case that arose in a histophysiological study which had shown that the degree of occlusion was related to the minimum number of nodes between the putative encoding sites. Three-dimensional reconstruction of the primary ending revealed that the terminals of one chain fibre were derived entirely from the first-order branch that supplied the bag1 fibre, including one terminal that was shared directly with the bag1 (sensory cross-terminal). The other first-order branch supplied the bag2 and remaining chain fibres as normal. The degree of occlusion seen during simultaneous stimulation of a dynamic beta axon and a static gamma axon indicated that the encoding sites were separated by both first-order branches. Schematic reconstruction of the motor innervation revealed that the static gamma axon was most unlikely to have supplied the chain fibre which shared sensory terminals with the bag1, but that these fibres also shared a motor input with histological characteristics of beta type. Ramp-frequency stimulation of the dynamic beta axon at constant length evoked a driving effect which persisted after fatiguing the extrafusal component and was therefore explicable on the basis of the observed pattern of motor innervation, though the identity of the axon could not be conclusively proved. Individually, instances of shared sensory terminals and motor input of bag1 and chain fibres are rare in the cat; their combination in a single spindle with correlated physiology is described here for the first time. The observation is considered in relation to the importance of dynamic and static segregation in motor control, since it may imply that there is a lower limit to the degree of segregation that the developmental programme can provide.


Combined antioxidants and anti-inflammatory therapies fail to attenuate the early and late phases of cyclodextrin-induced cochlear damage and hearing loss.
Niemann-Pick C1 (NPC1) is a fatal neurodegenerative disease caused by aberrant cholesterol metabolism. The progression of the disease can be slowed by removing excess cholesterol with high-doses of 2-hyroxypropyl-beta-cyclodextrin (HPβCD). Unfortunately, HPβCD causes hearing loss; the initial first phase involves a rapid destruction of outer hair cells (OHCs) while the second phase, occurring 4-6 weeks later, involves the destruction of inner hair cells (IHCs), pillar cells, collapse of the organ of Corti and spiral ganglion neuron degeneration. To determine whether the first and/or second phase of HPβCD-induced cochlear damage is linked, in part, to excess oxidative stress or neuroinflammation, rats were treated with a single-dose of 3000 mg/kg HPβCD alone or together with one of two combination therapies. Each combination therapy was administered from 2-days before to 6-weeks after the HPβCD treatment. Combination 1 consisted of minocycline, an antibiotic that suppresses neuroinflammation, and HK-2, a multifunctional redox modulator that suppresses oxidative stress. Combination 2 was comprised of minocycline plus N-acetyl cysteine (NAC), which upregulates glutathione, a potent antioxidant. To determine if either combination therapy could prevent HPβCD-induced hearing impairment and cochlear damage, distortion product otoacoustic emissions (DPOAE) were measured to assess OHC function and the cochlear compound action potential (CAP) was measured to assess the function of IHCs and auditory nerve fibers. Cochleograms were prepared to quantify the amount of OHC, IHC and pillar cell (PC) loss. HPβCD significantly reduced DPOAE and CAP amplitudes and caused significant OHC, IHC and OPC losses with losses greater in the high-frequency base of the cochlea than the apex. Neither minocycline + HK-2 (MIN+ HK-2) nor minocycline + NAC (MIN+NAC) prevented the loss of DPOAEs, CAPs, OHCs, IHCs or IPCs caused by HPβCD. These results suggest that oxidative stress and neuroinflammation are unlikely to play major roles in mediating the first or second phase of HPβCD-induced cochlear damage. Thus, HPβCD-induced ototoxicity must be mediated by some other unknown cell-death pathway possibly involving loss of trophic support from damaged support cells or disrupted cholesterol metabolism.


Targeted dietary approaches for the management of obesity and severe obesity in children and adolescents: A systematic review and meta-analysis.
There is a need for a detailed understanding of effective dietary interventions for children with obesity. This systematic review examined the effectiveness of diets of varying energy content as a component of weight treatment in children and adolescents with obesity, severe obesity and obesity-related comorbidity. A systematic search of six databases, from 2000 to 2021, for intervention studies of targeted dietary treatment for obesity in children aged 2-18 years identified 125 studies. Dietary interventions were grouped according to diet type and energy target. Risk of bias was assessed using the Effective Public Healthcare Panacea Project assessment tool. Meta-analysis examined change in body mass index (BMI) at intervention end. A broad array of diet types were effective at reducing BMI in children with obesity. When dietary types were considered by energy target, a gradient effect was observed. Very-low energy diets were most effective with a - 4.40 kg/m2 (n = 3; 95% CI -7.01 to -1.79). While dietary interventions with no specified energy target were ineffective, resulting in a BMI gain of +0.17 kg/m2 (n = 22; 95% CI 0.05 to 0.40). Practical definitions of dietary energy target in the management of obesity and severe obesity are urgently required to ensure treatment seeking children have timely access to efficacious interventions.


Leptin regulates chondrocyte differentiation and matrix maturation during endochondral ossification.
Leptin has been suggested to mediate a variety of actions, including bone development, via its ubiquitously expressed receptor (Ob-Rb). In this study, we investigated the role of leptin in endochondral ossification at the growth plate. The growth plates of wild-type and ob/ob mice were analyzed. Effects of leptin on chondrocyte gene expression, cell cycle, apoptosis and matrix mineralization were assessed using primary chondrocyte culture and the ATDC5 cell differentiation culture system. Immunohistochemistry and in situ hybridization showed that leptin was localized in prehypertrophic chondrocytes in normal mice and that Ob-Rb was localized in hypertrophic chondrocytes in normal and ob/ob mice. Growth plates of ob/ob mice were more fragile than those of wild-type mice in a mechanical test and were broken easily at the chondro-osseous junction. The growth plates of ob/ob mice showed disturbed columnar structure, decreased type X collagen expression, less organized collagen fibril arrangement, increased apoptosis and premature mineralization. Leptin administration in ob/ob mice led to an increase in femoral and humeral lengths and decrease in the proportional length of the calcified hypertrophic zone to the whole hypertrophic zone. In primary chondrocyte culture, the matrix mineralization in ob/ob chondrocytes was stronger than that of wild-type mice; this mineralization in both types of mice was abolished by the addition of exogenous leptin (10 ng/ml). During ATDC5 cell differentiation culture, exogenous leptin at a concentration of 1-10 ng/ml (equivalent to the normal serum concentration of leptin) altered type X collagen mRNA expression and suppressed apoptosis, cell growth and matrix calcification. In conclusion, we demonstrated that leptin modulates several events associated with terminal differentiation of chondrocytes. Our finding that the growth plates of ob/ob mice were fragile implies a disturbance in the differentiation/maturation process of growth plates due to depletion of leptin signaling in ob/ob mice. These findings suggest that peripheral leptin signaling plays an essential role in endochondral ossification at the growth plate.


Changes in the edentulous maxilla with ball and telescopic attachments of implant-retained mandibular overdentures: a 4-year retrospective study.
This retrospective study investigated the clinical and radiographic changes in the edentulous maxilla in patients with either ball or telescopic attachments of implantretained mandibular overdentures. Thirty-two completely edentulous patients (21 males and 11 females) received two implants in the canine region of the mandible. New maxillary complete dentures and mandibular overdentures were fabricated. Overdentures were connected to the implants either with ball (group 1, n = 16) or telescopic (group 2, n = 16) attachments. Retention and stability of the maxillary denture as well as mucosal changes of the maxillary ridge were recorded after 4 years of denturewearing. Traced rotational tomograms were used for measurements of maxillary alveolar bone loss. The proportional value between bone areas and areas of reference not subject to resorption was expressed as a ratio (R). Change in R immediately before (T0) and after 4 years (T4) of overdenture insertion was calculated for the anterior and posterior regions of the maxilla. After 4 years of denture-wearing, maxillary denture retention was significantly higher in group 1 than in group 2, while occurrence of flabby ridges was significantly higher in group 2 than in group 1. The change in R of the anterior region of the maxilla was significantly higher than the change in the posterior region in both groups. Group 2 showed significant anterior residual ridge resorption compared to group 1. Telescopic attachments for implant-retained mandibular overdentures are associated with increased maxillary ridge resorption and flabbiness, and decreased maxillary denture retention when compared to ball attachments.


Usefulness of risk stratification models for colorectal cancer based on fecal hemoglobin concentration and clinical risk factors.
We aimed to develop risk stratification models for advanced colorectal neoplasia (ACRN) and colorectal cancer (CRC) based on fecal hemoglobin (f-Hb) concentration and clinical risk factors. We reviewed screenees aged ≥50 years who underwent fecal immunochemical test (FIT) and colonoscopy and developed risk-scoring models for ACRN and CRC using logistic regression analysis. Participants were classified into low- (risk lower than that in FIT-negative individuals), intermediate- (risk higher than that in FIT-negative but lower than that in FIT-positive individuals), high- (risk similar to that in FIT-positive individuals), and very-high- (risk higher than that in FIT-positive individuals) risk groups. Of 3733 participants, 367 (9.8%) and 70 (1.9%) had ACRN and CRC, respectively. On multivariable analysis, age (ß = .043/y), former smoker (ß = .401), current smoker (ß = .841), diabetes (ß = .097), and square root of f-Hb concentration (ß = .071) were significantly associated with ACRN. In terms of CRC, age (ß = .035/y) and square root of f-Hb concentration (ß = .004) were associated factors. After point assignments based on the regression coefficient, we could classify screenees as low-, intermediate-, high-, and very-high-risk groups. ACRN was identified in 2.9%, 5.3%, 16.2%, and 35.7% of screenees in the low-, intermediate-, high-, and very-high-risk groups, respectively. CRC was identified in .1%, .5%, 3.9%, and 11.1% of screenees in the low-, intermediate-, high-, and very-high-risk groups, respectively. The proposed models can effectively stratify the risk for ACRN and CRC and provide accurate information on this risk in individuals who undergo FIT.


A flower-like hybrid material composed of Fe3O4, graphene oxide and CdSe nanodots for magnetic solid phase extraction of ibuprofen prior to its quantification by HPLC detection.
A flower-like Fe3O4/GO/CdSe nanodot magnetic hybrid material was produced and applied to magnetic solid-phase extraction of ibuprofen from pharmaceuticals, water, and urine samples. The material was characterized by X-ray diffraction, Raman spectroscopy and field emission scanning electron microscopy and SEM-EDX. The pH value, volume of sample solution, amount of sorbent, type and volume of elution solvent and extraction time were optimized. Following elution with acetone, ibuprofen was quantified by HPLC-DAD detection. The recoveries of ibuprofen from spiked real samples ranged between 87 and 109%, and the intra-day and inter-day relative standard deviations from 1.25 to 3.02%. The limit of detection, limit of quantification and preconcentration factor are 0.36 ng·mL-1,1.20 ng·mL-1 and 150, respectively. Graphical abstract Schematic representation of the combination of flower-like Fe3O4/GO/CdSe nanodot-based magnetic solid phase extraction (MSPE) and high-performance liquid chromatography (HPLC) procedure for the extraction and analysis of ibuprofen in pharmaceuticals, water, and urine samples.


Targeting stromal cell sialylation reverses T cell-mediated immunosuppression in the tumor microenvironment.
Immunosuppressive tumor microenvironments (TMEs) reduce the effectiveness of immune responses in cancer. Mesenchymal stromal cells (MSCs), precursors to cancer-associated fibroblasts (CAFs), promote tumor progression by enhancing immune cell suppression in colorectal cancer (CRC). Hyper-sialylation of glycans promotes immune evasion in cancer through binding of sialic acids to their receptors, Siglecs, expressed on immune cells, which results in inhibition of effector functions. The role of sialylation in shaping MSC/CAF immunosuppression in the TME is not well characterized. In this study, we show that tumor-conditioned stromal cells have increased sialyltransferase expression, α2,3/6-linked sialic acid, and Siglec ligands. Tumor-conditioned stromal cells and CAFs induce exhausted immunomodulatory CD8+ PD1+ and CD8+ Siglec-7+/Siglec-9+ T cell phenotypes. In vivo, targeting stromal cell sialylation reverses stromal cell-mediated immunosuppression, as shown by infiltration of CD25 and granzyme B-expressing CD8+ T cells in the tumor and draining lymph node. Targeting stromal cell sialylation may overcome immunosuppression in the CRC TME.


Viral skin infections: diagnosis and treatment considerations.
Skin lesions are prominent features of many viral diseases. In some instances, characteristic skin lesions suggest a specific viral illness, the diagnosis of which can be quickly established by appropriate procedures. In addition to clinical manifestations, laboratory methods including virus isolation are used to diagnose viral infections. In viral diseases, prophylaxis has proved more successful than the specific treatment of established infection. However, recent progress in molecular biology has facilitated the development of new vaccines and new drugs to treat viral infections.


Level of linoleic acid in pericardial lipids of Charollais cows with and without ovarian cysts.
The content of linoleic acid, a precursor of prostaglandins, in pericardial lipid store is higher in cows with normal ovaries than in cows with cystic ovaries.


Rectorite-intercalated nanoparticles for improving controlled release of doxorubicin hydrochloride.
Controlled release of drugs has been widely researched in biomedical area. Nanoparticles (NPs) as ideal drug carriers are often used to facilitate improvements in the therapeutic index of drugs. In this study, natural polymers carboxymethyl chitosan (CMC) and lysozyme (LY) were mixed to prepare CMC-LY NPs by electrostatic self-assembly interactions. In addition, layered silicate rectorite (REC) was introduced into NPs to explore the effect on the encapsulation efficacy and controlled release of doxorubicin hydrochloride (DOX). It was confirmed that the average size of NPs increased with the addition of REC, and the interlayer distance of REC in NPs was enlarged because of the intercalation with polymer chains. Besides, the encapsulation efficiency and loading capacity of DOX in NPs increased markedly with the accretion of REC. The incorporation of REC into NPs could reduce the initial burst release and prolong the therapeutic time. Such results suggest that the REC-intercalated NPs are promising anticancer drug carriers for efficient cancer therapy.


Carboplatin-paclitaxel compared to Carboplatin-Paclitaxel-Bevacizumab in advanced or recurrent endometrial cancer: MITO END-2 - A randomized phase II trial.
Increased Vascular Endothelial Growth Factor Receptor (VEGF) expression in endometrial cancer (EC) is associated with a poor prognosis. Preliminary clinical data reported Bevacizumab effectiveness in EC both as single agent and in combination with chemotherapy. In a phase II trial, patients with advanced (FIGO stage III-IV) or recurrent EC were randomized to receive Carboplatin-Paclitaxel standard dose for 6-8 cycles vs Carboplatin-Paclitaxel and Bevacizumab 15 mg/kg in combination with chemotherapy and maintenance until disease progression or unacceptable toxicity. The primary endpoint was progression free survival (PFS). 108 patients were randomized; PFS (10.5 vs 13.7 months, HR 0.84 p = 0.43), overall response rate (ORR 53.1% vs 74.4%) and overall survival (OS) (29.7 vs 40.0 months, HR 0.71 p = 0.24) resulted in a non-significant increase in Bevacizumab treated patients. The PFS increase became significant when an exploratory analysis with the Breslow test was used. Moreover, patients treated with Bevacizumab experienced a significant increase in 6-month disease control rate (70.4% vs 90.7%). Cardiovascular events were more frequent in the experimental arm ("de novo" grade ≥2 hypertension 21% vs 0% and grade ≥2 thromboembolic events 11% vs 2% in the Bevacizumab vs standard treatment arm, respectively). Bevacizumab combined with chemotherapy in the treatment of advanced/recurrent EC failed to demonstrate a significant increase in PFS in the MITO END-2 trial. Nevertheless, these preliminary data suggests some effectiveness of the antiangiogenic agent which merits further exploration in a larger population with a better molecular characterization.


Effects of lactic acid infusions and pH on cerebral blood flow and metabolism.
To determine the effects of lactic acidemia versus lactate on CBF, we infused lactic acid, either buffered with NaOH (L + NaOH) or with added NaCl (L + NaCl), to attain similar osmolalities in 18 piglets. CBF (microsphere technique), pH, blood gases, plasma osmolality, and cerebral arteriovenous differences of O2 content and lactic acid concentrations were measured prior to, at 30 min of a lactic acid infusion, and 15 and 90 min after completion of the infusion. Control arterial pH was comparable between groups (7.50 +/- 0.02 vs. 7.49 +/- 0.02, X +/- SE); during and following L + NaCl and L + NaOH, values were (p less than 0.05) 7.09 +/- 0.03, 7.35 +/- 0.02, and 7.46 +/- 0.02 vs. 7.58 +/- 0.03, 7.61 +/- 0.01, and 7.57 +/- 0.03, respectively. PaCO2 remained unchanged and osmolality rose by 15% in both groups during infusions and persisted throughout the study period. For L + NaCl piglets, CBF (ml/min.100 g) rose from 136 +/- 15 to 198 +/- 26 (p less than 0.05) at 30 min of infusion and remained elevated at 201 +/- 25 and 207 +/- 28 at 15 and 90 min following the infusion, respectively. Similarly, for L + NaOH piglets, CBF rose from 130 +/- 25 to 196 +/- 31 (p less than 0.05) with the infusion and was 174 +/- 17 and 166 +/- 21 at 15 and 90 min afterward, respectively. Although lactic acid infusion increases CBF, the associated metabolic acidemia is not responsible for changes in CBF.


Improvement in molecule exchange efficiency in Gibbs ensemble Monte Carlo: development and implementation of the continuous fractional component move.
The continuous fractional component Monte Carlo (CFC MC) move (J Chem Theory Comput, 2007, 3, 1451) is extended to the Gibbs ensemble. The algorithm is validated against conventional simulations for the Lennard Jones fluid and a flexible water model. The method is also used to compute the vapor-liquid coexistence densities of a model for SO(2). The CFC molecule exchange move relies on the gradual insertion and deletion of molecules in conjunction with a self-adapting bias potential. As a result, the method does not require the formation of spontaneous voids in the dense fluid phase to be successful, leading to molecule exchange acceptance probabilities that are nearly independent of temperature. For example, over 1% of the vapor-liquid molecule exchange moves are successful for water at 280 K, whereas advanced rotational and configurational bias methods have success rates of less than 0.03%. The CFC move can be combined with other Monte Carlo moves to enable efficient simulation of dense strongly associating fluids that are to this point infeasible to model with standard methods.


Involvement of adenosine A2B receptor in radiation-induced translocation of epidermal growth factor receptor and DNA damage response leading to radioresistance in human lung cancer cells.
Adenosine receptors are involved in tumor growth, progression, and response to therapy. Among them, A2B receptor is highly expressed in various tumors. Furthermore, ionizing radiation induces translocation of epidermal growth factor receptor (EGFR), which promotes DNA repair and contributes to radioresistance. We hypothesized that A2B receptor might be involved in the translocation of EGFR. We investigated whether A2B receptor is involved in EGFR translocation and DNA damage response (γH2AX/53BP1 focus formation) of lung cancer cells by means of immunofluorescence studies. Radiosensitivity was evaluated by colony formation assay after γ-irradiation. A2B receptor was expressed at higher levels in cancer cells than in normal cells. A2B receptor antagonist treatment or A2B receptor knockdown suppressed EGFR translocation, γH2AX/53BP1 focus formation, and colony formation of lung cancer cell lines A549, calu-6 and NCI-H446, compared with a normal cell line (beas-2b). γ-Irradiation-induced phosphorylation of src and EGFR was also attenuated by suppression of A2B receptor expression. Activation of A2B receptor mediates γ-radiation-induced translocation of EGFR and phosphorylation of src and EGFR, thereby promoting recovery of irradiated lung cancer cells from DNA damage. Our results indicate that A2B receptors contribute to radiation resistance in a cancer-cell-specific manner, and may be a promising target for radiosensitizers in cancer radiotherapy.


Isolation of a rat immune response gene identical to an alleged mouse a class II beta-chain pseudogene.
A human HLA-DQ beta-chain cDNA was used as a probe to identify and isolate a rat major histocompatibility antigen beta-chain gene from a genomic library constructed in the vector lambda Charon 28 using Wistar rat DNA (RT1u). The isolated exon of the rat gene (RT1.B beta 2) encoding a beta-chain second domain was found to share 93% nucleotide homology with a mouse A beta 2 exon. Although the genomic organization of this gene is consistent with the hypothesis that it represents a pseudogene, the remarkable preservation of a specific sequence favors the view that this class II antigen beta-chain gene has retained its coding function.


Concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem II.
We describe a concentric-flow electrokinetic injector for efficiently delivering microcrystals for serial femtosecond X-ray crystallography analysis that enables studies of challenging biological systems in their unadulterated mother liquor. We used the injector to analyze microcrystals of Geobacillus stearothermophilus thermolysin (2.2-Å structure), Thermosynechococcus elongatus photosystem II (<3-Å diffraction) and Thermus thermophilus small ribosomal subunit bound to the antibiotic paromomycin at ambient temperature (3.4-Å structure).


Th2 and eosinophil responses suppress inflammatory arthritis.
Th2-eosinophil immune responses are well known for mediating host defence against helminths. Herein we describe a function of Th2-eosinophil responses in counteracting the development of arthritis. In two independent models of arthritis, Nippostrongylus brasiliensis infection leads to Th2 and eosinophil accumulation in the joints associated with robust inhibition of arthritis and protection from bone loss. Mechanistically, this protective effect is dependent on IL-4/IL-13-induced STAT6 pathway. Furthermore, we show that eosinophils play a central role in the modulation of arthritis probably through the increase of anti-inflammatory macrophages into arthritic joints. The presence of these pathways in human disease is confirmed by detection of GATA3-positive cells and eosinophils in the joints of rheumatoid arthritis patients. Taken together, these results demonstrate that eosinophils and helminth-induced activation of the Th2 pathway axis effectively mitigate the course of inflammatory arthritis.


Treatment of pica in an adult disabled by autism by differential reinforcement of incompatible behavior.
The usefulness of differential reinforcement of incompatible behavior was examined in the treatment of pica in a young man severely disabled by autism in a nonsheltered place of employment. The results indicated that a reduction in pica could be achieved by differential reinforcement of other behavior (DRO). Behaviors which were reinforced included remaining in this assigned location, keeping his hands on his work, working quickly and keeping his mouth clear. Reinforcements included favorite drinks, snacks, activities and praise. Treatment procedures were implemented by direct care paraprofessional staff. Withdrawal of treatment resulted in an increase in pica, with renewed reduction on reinstatement of the reinforcement schedule.


A unifying parameter to describe the clinical mechanics of hydrogel contact lenses.
We studied the relation of postlens squeeze pressure to the clinical fit of hydrogel contact lenses. This relation was statistically significant (p less than 0.01) for a variety of contact lenses currently prescribed over a range of thickness, water content, back central optic radius, diameter, and back vertex power. The squeeze pressure provided a useful parameter to describe and compare the clinical fit of hydrogel contact lenses and a model with which the fit of a lens to an eye could be predicted. There was virtually no movement of the contact lenses across the eye when the squeeze pressure was more than -14 mm H2O (-1370 dynes/cm2). However, the mechanics of the lens motion when the squeeze pressure was less than this critical pressure appeared to be related to a more complicated combination of the squeeze pressure and the other forces in the eye-lens system.


Serologic evidence of Venezuelan equine encephalitis in some wild and domestic populations of southern Texas.
More than 2,500 sera from approximately 30 wild and domestic species in southern Texas were tested for neutralizing antibodies to Venezuelan equine encephalitis (VEE). Virus isolations were also attempted from blood and tissue samples of many of the wild specimens. VEE neutralizing substances were present in a variety of species collected prior to the 1971 epizootic, suggesting that VEE was present and perhaps enzootic in this area before the recent epizootic. Serologic results of this study suggest that deer (Odocoileus virginianus) and feral swine (Sus scrofa) may serve as good indicators or sentinels of VEE transmission. The reservoir of VEE was not established, but results of the study suggest that a number of species or a combination of animal host populations including deer, feral swine, and peccaries (Pecari angulatus) may be involved in the eizootiologyof VEE in southern Texas.


Individualizing drug dosage by using a random intercept linear model.
An algorithm for drug dosage individualization is proposed. The algorithm assumes a random intercept linear model for the log of trough-plasma-concentration-to-dosage ratio of the drug at steady-state, and aims at determining an optimum dosage for producing a trough steady-state plasma concentration within a target concentration range. The minimum number of algorithm steps necessary to find the optimum dosage is computed. Computations are illustrated for clozapine, an antipsychotic drug used to treat patients with severe schizophrenia.


Comparative study of equine mesenchymal stem cells from healthy and injured synovial tissues: an in vitro assessment.
Bone marrow and adipose tissues are known sources of mesenchymal stem cells (MSCs) in horses; however, synovial tissues might be a promising alternative. The aim of this study was to evaluate phenotypic characteristics and differentiation potential of equine MSCs from synovial fluid (SF) and synovial membrane (SM) of healthy joints (SF-H and SM-H), joints with osteoarthritis (SF-OA and SM-OA) and joints with osteochondritis dissecans (SF-OCD and SM-OCD) to determine the most suitable synovial source for an allogeneic therapy cell bank. Expression of the markers CD90, CD105, CD44, and CD34 in SF-H, SM-H, SF-OA, SM-OA, SF-OCD and SM-OCD was verified by flow cytometry, and expression of cytokeratin, vimentin, PGP 9.5, PCNA, lysozyme, nanog, and Oct4 was verified by immunocytochemistry. MSCs were cultured and evaluated for their chondrogenic, osteogenic and adipogenic differentiation potential. Final quantification of extracellular matrix and mineralized matrix was determined using AxioVision software. A tumorigenicity test was conducted in Balb-C(nu/nu) mice to verify the safety of the MSCs from these sources. Cultured cells from SF and SM exhibited fibroblastoid morphology and the ability to adhere to plastic. The time elapsed between primary culture and the third passage was approximately 73 days for SF-H, 89 days for SF-OCD, 60 days for SF-OA, 68 days for SM-H, 57 days for SM-OCD and 54 days for SM-OA. The doubling time for SF-OCD was higher than that for other cells at the first passage (P < 0.05). MSCs from synovial tissues showed positive expression of the markers CD90, CD44, lysozyme, PGP 9.5, PCNA and vimentin and were able to differentiate into chondrogenic (21 days) and osteogenic (21 days) lineages, and, although poorly, into adipogenic lineages (14 days). The areas staining positive for extracellular matrix in the SF-H and SM-H groups were larger than those in the SF-OA and SM-OA groups (P < 0.05). The positive mineralized matrix area in the SF-H group was larger than those in all the other groups (P < 0.05). The studied cells exhibited no tumorigenic effects. SF and SM are viable sources of equine MSCs. All sources studied provide suitable MSCs for an allogeneic therapy cell bank; nevertheless, MSCs from healthy joints may be preferable for cell banking purposes because they exhibit better chondrogenic differentiation capacity.