Fabrication process development and basic evaluation of eggshell-based column packing material for reversed-phase preparative separation.
Purification of basic drugs in reversed-phase mode is often difficult, mainly due to adsorption of positively charged compounds to the silica gel-based stationary phase. Since this adsorption can be suppressed under alkaline condition, columns with alkali-resistance are required. In addition, compounds with acid-sensitive structures are sometimes degraded during separation on silica gel-based columns which exhibit acidity due to their surface structure. We prepared an alkali-resistant reversed-phase packing material, Eggshell-PMAcO based on eggshells modified with an amphiphilic copolymer, poly(maleic acid-alt-1-octadecene) (PMAcO). The height equivalent to a theoretical plate (HETP) of the Eggshell-PMAcO column was improved by surface treatment with ammonium acetate buffer (900 mM, pH = 3.7), which is an inexpensive reagent, and the retention behavior for hydrophobic compounds was compared to a typical ODS column based on silica gel, resulting in sufficient selectivity of the eggshell-based column for hydrophobic compounds, as indicated by the ratio of retention factors of pentylbenzene and butylbenzene (Eggshell-PMAcO column: 1.55, ODS column: 1.65). Column temperature-dependent retention behavior of naphthalene was investigated in the temperature range from 25 °C to 45 °C, followed by the calculation of thermodynamic parameters. There was little difference in the standard molar enthalpy (Eggshell-PMAcO: -19.6 kJ/mol, ODS: -21.7 kJ/mol). The absolute value of the standard free Gibbs energy for the Eggshell-PMAcO column was much smaller than that of the ODS column (Eggshell-PMAcO: -0.284 kJ/mol, ODS: -13.0 kJ/mol), indicating that the Eggshell-PMAcO column had a weaker retention strength for naphthalene than the ODS column mainly due to the large difference in the standard molar entropy (Eggshell-PMAcO: -64.9 J/mol K, ODS column: -29.2 J/mol K). The retention capacities for imipramine under neutral (water/methanol) and alkaline (0.1% triethylamine water/methanol) conditions were 0.2 mg and 5 mg, respectively, based on injection mass-dependent HETP, retention factor and symmetry factor. Finally, the prepared column was applied to the purification of a building block for nucleic acid drugs. This study demonstrated that reversed-phase columns, which can be fabricated from eggshells and an amphiphilic copolymer in an inexpensive and eco-friendly way, have the ability to purify basic compounds and acid-sensitive compounds.


Nanostructured GaAs solar cells via metal-assisted chemical etching of emitter layers.
GaAs solar cells with nanostructured emitter layers were fabricated via metal-assisted chemical etching. Au nanoparticles produced via thermal treatment of Au thin films were used as etch catalysts to texture an emitter surface with nanohole structures. Epi-wafers with emitter layers 0.5, 1.0, and 1.5 um in thickness were directly textured and a window layer removal process was performed before metal catalyst deposition. A nanohole-textured emitter layer provides effective light trapping capabilities, reducing the surface reflection of a textured solar cell by 11.0%. However, because the nanostructures have high surface area to volume ratios and large numbers of defects, various photovoltaic properties were diminished by high recombination losses. Thus, we have studied the application of nanohole structures to GaAs emitter solar cells and investigated the cells' antireflection and photovoltaic properties as a function of the nanohole structure and emitter thickness. Due to decreased surface reflection and improved shunt resistance, the solar cell efficiency increased from 4.25% for non-textured solar cells to 7.15% for solar cells textured for 5 min.


Daily intake of a formulated tomato drink affects carotenoid plasma and lymphocyte concentrations and improves cellular antioxidant protection.
The salutary characteristics of the tomato are normally related to its content of carotenoids, especially lycopene, and other antioxidants. Our purpose was to verify whether the daily intake of a beverage prototype called Lyc-o-Mato((R)) containing a natural tomato extract (Lyc-o-Mato((R)) oleoresin 6 %) was able to modify plasma and lymphocyte carotenoid concentrations, particularly those of lycopene, phytoene, phytofluene and beta-carotene, and to evaluate whether this intake was sufficient to improve protection against DNA damage in lymphocytes. In a double-blind, cross-over study, twenty-six healthy subjects consumed 250 ml of the drink daily, providing about 6 mg lycopene, 4 mg phytoene, 3 mg phytofluene, 1 mg beta-carotene and 1.8 mg alpha-tocopherol, or a placebo drink. Treatments were separated by a wash-out period. Plasma and lymphocyte carotenoid and alpha-tocopherol concentrations were determined by HPLC, and DNA damage by the comet assay. After 26 d of consumption of the drink, plasma carotenoid levels increased significantly: concentrations of lycopene were 1.7-fold higher (P<0.0001); of phytofluene were 1.6-fold higher (P<0.0001); of phytoene were doubled (P<0.0005); of beta-carotene were 1.3-fold higher (P<0.05). Lymphocyte carotenoid concentrations also increased significantly: that of lycopene doubled (P<0.001); that of phytofluene was 1.8-fold higher (P<0.005); that of phytoene was 2.6-fold higher (P<0.005); that of beta-carotene was 1.5-fold higher (P<0.01). In contrast, the alpha-tocopherol concentration remained nearly constant. The intake of the tomato drink significantly reduced (by about 42 %) DNA damage (P<0.0001) in lymphocytes subjected to oxidative stress. In conclusion, the present study supports the fact that a low intake of carotenoids from tomato products improves cell antioxidant protection.


Perioperative buffered versus non-buffered fluid administration for surgery in adults.
Perioperative fluid therapy influences clinical outcomes following major surgery. Fluid preparations may be based on a simple non-buffered salt solution, such as normal saline, or may be modified with bicarbonate or bicarbonate precursor buffers, such as maleate, gluconate, lactate or acetate, to better reflect the human physiological state. These latter fluids have theoretical advantages over normal saline in preventing hyperchloraemic acidosis. A number of clinical studies have now compared fluid preparations with and without a buffer to achieve a balanced electrolyte solution for perioperative fluid resuscitation. To review the safety and efficacy of perioperative administration of buffered versus non-buffered fluids for plasma volume expansion or maintenance in adult patients undergoing surgery. We electronically searched the Cochrane Central Register of Controlled Trials (CENTRAL) (2011, Issue 4), MEDLINE (1966 to May 2011), EMBASE (1980 to May 2011), and CINAHL (1982 to May 2011). We handsearched conference abstracts and where possible, contacted leaders in the field. We only included randomized trials of buffered versus non-buffered intravenous fluids for perioperative fluid resuscitation. The trials with other forms of comparisons such as crystalloids versus colloids and colloids versus different colloids were excluded. We also excluded trials using hypertonic fluids and dextrose-based fluids. Two authors independently extracted data and assessed the methodological quality of clinical trials. We resolved any disagreements by discussion. We contacted the trial authors to provide additional information where appropriate. We presented pooled estimates of the dichotomous outcomes as odds ratios (OR) and on continuous outcomes as mean differences, with 95% confidence intervals (CI). We analysed data on Review Manager 5.1 using fixed-effect models, and when heterogeneity was high (I² > 40%) random-effect models were used. We identified 14 publications reporting 13 trials or comparisons with a total of 706 participants. For many of the outcomes reported, there was significant clinical and statistical heterogeneity. The primary outcome of mortality at any time was reported in only three studies with a total of 267 patients. The mortality rate was 2.9% for the buffered fluids group and 1.5% for the non-buffered fluids group but this difference was not statistically significant. The Peto OR was 1.85 (95% CI 0.37 to 9.33, P = 0.45, I(2)= 0%). Organ dysfunction was only presented for renal impairment. There was no difference in renal insufficiency leading to renal replacement therapy between the buffered and non-buffered groups (OR 0.61, 95% CI 0.23 to 1.63, P = 0.32, I(2) = 0%). Markers of organ system failure as assessed by urine output, creatinine and its variables (for renal function), PaC0(2) (respiratory function) and postoperative nausea and vomiting (gastro-intestinal function) showed a statistically significant difference only in PaC0(2) levels. The mean difference was 1.18 with lower PaC0(2) levels in the non-buffered fluid group (95% CI 0.09 to 2.28, P = 0.03, I(2) = 0%) compared to the buffered fluid group.There was no difference in intraoperative blood loss nor the volumes of intraoperative red cell or fresh frozen plasma transfused between groups. There was an increase in platelet transfusion in the non-buffered group which was statistically significant after analysing the transformed data (log transformation because the data were highly skewed).A number of metabolic differences were noted. There was a difference in postoperative pH of 0.06 units, lower in the non-buffered fluid group (95% CI 0.04 to 0.08, P < 0.00001, I(2) = 74%). However, this difference was not maintained on postoperative day one. The non-buffered fluid group also had significantly greater base deficit, serum sodium and chloride levels.There was no difference demonstrated in length of hospital stay and no data were reported on cost or quality of life. The administration of buffered fluids to adult patients during surgery is equally safe and effective as the administration of non-buffered saline-based fluids. The use of buffered fluids is associated with less metabolic derangement, in particular hyperchloraemia and metabolic acidosis. Larger studies are needed to assess robust outcomes such as mortality.


Architecture of the mammalian pituitary cholinergic system with observations on a putative blood acetylcholine sensor.
Acetylcholine (ACh) plays an important role in pituitary gland function. Little is known, however, about the source and trajectory of pituitary ACh, the location of pituitary cholinergic receptors, and the pathways along which the release of pituitary ACh is controlled. Therefore choline acetyltransferase (CHAT) immunoreactive profiles have been investigated in the rat median eminence and pituitary. Furthermore, both muscarinic- (mAChRp-L) and nicotinic receptor proteinlike (nAChRp-L) immunoreactivity have been examined in the rat, rabbit, and cat pituitary. The results have demonstrated that the rat pituitary ChAT network is composed of neurons in the hypothalamic arcuate nucleus and a great number of terminals in the median eminence. In the pituitary, ChAT immunolabeled profiles were virtually absent. This suggests that much of the ACh acting on pituitary cells is released as a humoral factor from the median eminence. All the examined animals expressed mAChRp-L immunostained endocrine cells in the intermediate lobe. Apart from this, marked species differences in AChRp-L immunolabeled profiles have been found. In addition, strong mAChRp-L immunoreactive rod to cone-shaped bodies were detected associated with blood vessels of the anterior and intermediate lobes in the rat and rabbit, but not in the cat. The immunolabeling was present in particles on the body plasma membrane. These characteristics suggest that the function of these structures might be to sense pituitary blood ACh levels. Consequently the name blood acetylcholine reading bodies (BARBs) was adopted to indicate these structures. It is proposed that the BARBs may play a role in the feedback control of ACh release from the median eminence.


Aberrant expression of miR-9/9* in myeloid progenitors inhibits neutrophil differentiation by post-transcriptional regulation of ERG.
Aberrant post-transcriptional regulation by microRNAs (miRNAs) has been shown to be involved in the pathogenesis of acute myeloid leukemia (AML). In a previous study, we performed a large functional screen using a retroviral barcoded miRNA expression library. Here, we report that overexpression of miR-9/9* in myeloid 32D cell line (32D-miR-9/9*) had profound impact on granulocyte colony-stimulating factor-induced differentiation. Further in vitro studies showed that enforced expression of miR-9/9* blocked normal neutrophil development in 32D and in primary murine lineage-negative bone marrow cells. We examined the expression of miR-9/9* in a cohort of 647 primary human AMLs. In most cases, miR-9 and miR-9* were significantly upregulated and their expression levels varied according to AML subtype, with the highest expression in MLL-related leukemias harboring 11q23 abnormalities and the lowest expression in AML cases with t(8;21) and biallelic mutations in CEBPA. Gene expression profiling of AMLs with high expression of miR-9/9* and 32D-miR-9/9* identified ETS-related gene (Erg) as the only common potential target. Upregulation of ERG in 32D cells rescued miR-9/9*-induced block in neutrophil differentiation. Taken together, this study demonstrates that miR-9/9* are aberrantly expressed in most of AML cases and interfere with normal neutrophil differentiation by downregulation of ERG.


The effects of different patterns of muscle activity on capillary density, mechanical properties and structure of slow and fast rabbit muscles.
When rabbit fast muscles were chronically stimulated at a frequency naturally occurring in nerves to slow muscles (10 Hz), there was a transformation towards a slow muscle type such as an increase of capillary density, increased activity of the oxidative enzyme, succinic dehydrogenase, and a decrease of muscle fibre diameters. After 28 days the intensity and distribution of SDH and the capillary density were similar to those of soleus. The increases in capillary density preceded the changes in activity of SDH; there was a significantly greater capillary/muscle fibre ratio and number of capillaries/mm2 in muscles stimulated for only 4 days at which time no change could be detected in SDH. These changes were induced by slow frequency stimulation only, and not by an overall increase of activity. Stimulation of fast muscles for 4 days at a higher frequency naturally occurring in the nerves to fast muscles (short bursts of tetani), with the same total number of stimuli as that used in slow frequency stimulation did not produce any changes in capillary density, activity of SDH or contraction times. No changes were observed in either fast or slow muscles stimulated with short bursts of tetani (and lower total number of impulses) up to 28 days. Activation of fast muscles at 5 Hz continuously or 10 Hz intermittently also caused an increase in capillary density. It is therefore concluded that only low frequency activation of fast muscles brings about a transformation of the muscle fibres towards a slow type and that the first noticeable change is an increase in the capillary density.


Efficacy of oral compared with intramuscular vitamin B-12 supplementation after Roux-en-Y gastric bypass: a randomized controlled trial.
After Roux-en-Y gastric bypass (RYGB), patients often develop a vitamin B-12 deficiency. Our objective was to investigate whether oral supplementation increases and normalizes low vitamin B-12 concentrations (vitamin B-12 > 200 pmol/L) in RYGB patients as compared to intramuscular injections. A randomized controlled trial in RYGB patients with subnormal serum B-12 concentrations was performed. One group (IM B-12) received bimonthly intramuscular hydroxocobalamin injections (2000 µg as loading dose and 1000 µg at follow-up) for 6 mo. The second group (oral B-12) received daily doses of oral methylcobalamin (1000 µg). Serum vitamin B-12 was determined at baseline (T0) and at 2 (T1), 4 (T2), and 6 mo (T3) after start of treatment. Concentrations of the secondary markers methylmalonic acid (MMA) and homocysteine (Hcy) were measured at T0 and T3. Fifty patients were included and randomized, 27 in IM B-12 and 23 in oral B-12. The median vitamin B-12 concentration at T0 was 175 pmol/L (range: 114-196 pmol/L) for IM B-12 and 167 pmol/L (range: 129-199 pmol/L) for oral B-12. Vitamin B-12 normalized in all individuals, and there was no significant difference in vitamin B-12 between the two groups. MMA and Hcy concentrations decreased significantly after 6 mo within each group (P < 0.001 and P < 0.001 for MMA and P = 0.03 and P = 0.045 for Hcy, respectively). There was no significant difference between the groups at 6 mo for both MMA and Hcy (P = 0.53 and P = 0.79). The efficacy of oral vitamin B-12 supplementation was similar to that of hydroxocobalamin injections in the present study. Oral supplementation can be used as an alternative to hydroxocobalamin injections to treat RYGB patients with low values of serum vitamin B-12. This trial was registered at clinicaltrials.gov as NCT02270749.


The cancer biomarker osteopontin: combination with other markers.
Osteopontin (OPN) is a biomarker for cancer progression and prognosis in multiple tumor types, however, it has not yet found entry into clinical diagnostics. In recent years, there has been an increasing recognition that marker combinations may have better diagnostic or prognostic potential than individual molecules. While various studies have analyzed OPN in conjunction with other markers, a comprehensive review of their published results is still lacking. OPN in tumor tissue or in the blood has been investigated in conjunction with a broad range of other markers in diverse types of cancer. OPN has been combined with cancer-specific markers, functionally converging biomolecules (angiogenesis, motility/adhesion, extracellular matrix, bone) and synergizing biomolecules (fibrinolytic system, calcium homeostatic proteins, squamous cell carcinoma antigen, NF-κB pathways, proteases). Clinical parameters of interest have been cancer detection, assessment of progression, and prognosis/treatment response. Some marker combinations with OPN are promising for detection, diagnosis or prognosis in various types of cancer. Yet, surprisingly, in many cases, the published results are conflicting, and no clear metrics have been developed for utilizing marker combinations in clinical decision making. With the current intense interest in multiplex marker panels, additional, large-scale studies are needed to realize the full diagnostic and prognostic potential of OPN.


Reactive-Accelerated-Aging Testing of Thinned Tissue-Engineered Electronic Nerve Interfaces.
Tissue responses can cause a significant reduction in the performance of microelectrode-based devices implanted into neural tissue. Since the reduction of the thickness of implants has been shown to reduce tissue response, in this work we report on our effects to reduce the thickness of our tissue-engineered-electronic-nerve-interface (TEENI) devices and characterize their long-term reliability in a harsh environment. We were able to reduce the thickness of the TEENI threads that are to be located in nerve tissue from ~10 μm to ~2.5 μm in total thickness. To maintain the handleability needed during the assembly of the TEENI device into the hydrogel-based scaffold, we maintained full thickness elsewhere in the TEENI device and added support rails. During longitudinal reactive-accelerated-aging (RAA) experiments performed over 6 days and at 67°C, which corresponds to ~48 days in tissue, we observed that some channels maintain a stable impedance and others do not. Although analyses performed using a scanning electron microscope could clearly reveal delamination in some channels that exhibited large changes in impedance, it did not always correlate. Some channels with significant changes in impedance did not exhibit any observable delamination. Additional work is needed to study the relationship between changes in impedance and structural changes in the device, with the goal of improving device design to achieve longer-lasting devices.


Mycotic aneurysms in transplant patients.
Eight cases of mycotic aneurysm occurred in seven transplant patients. Perinephric wound infection involving the iliac arteries was the cause of at least seven aneurysms. Simple ligation of the iliac artery proximal and distal to the aneurysm without grafting was effective therapy in all but one patient with an aortic aneurysm who required an axillofemoral bypass graft prior to excision of the aneurysm. There were no instances of ischemia to the involved extremities, and only two patients died as a result of the aneurysm. This dangerous complication can be avoided by preventing wound infections. In established perinephric abscesses, the kidney should be removed and the wound kept as clean as possible in order to prevent this complication. Arteriograms may be useful in the early detection of these aneurysms, before they rupture.


Genome-Wide Datasets of Chicories (Cichorium intybus L.) for Marker-Assisted Crop Breeding Applications: A Systematic Review and Meta-Analysis.
Cichorium intybus L. is the most economically important species of its genus and among the most important of the Asteraceae family. In chicory, many linkage maps have been produced, several sets of mapped and unmapped markers have been developed, and dozens of genes linked to traits of agronomic interest have been investigated. This treasure trove of information, properly cataloged and organized, is of pivotal importance for the development of superior commercial products with valuable agronomic potential in terms of yield and quality, including reduced bitter taste and increased inulin production, as well as resistance or tolerance to pathogens and resilience to environmental stresses. For this reason, a systematic review was conducted based on the scientific literature published in chicory during 1980-2023. Based on the results obtained from the meta-analysis, we created two consensus maps capable of supporting marker-assisted breeding (MAB) and marker-assisted selection (MAS) programs. By taking advantage of the recently released genome of C. intybus, we built a 639 molecular marker-based consensus map collecting all the available mapped and unmapped SNP and SSR loci available for this species. In the following section, after summarizing and discussing all the genes investigated in chicory and related to traits of interest such as reproductive barriers, sesquiterpene lactone biosynthesis, inulin metabolism and stress response, we produced a second map encompassing 64 loci that could be useful for MAS purposes. With the advent of omics technologies, molecular data chaos (namely, the situation where the amount of molecular data is so complex and unmanageable that their use becomes challenging) is becoming far from a negligible issue. In this review, we have therefore tried to contribute by standardizing and organizing the molecular data produced thus far in chicory to facilitate the work of breeders.


Intravenous urography in the emergency department: when do we need it?
Intravenous urography (IVU) is a useful radiographic study in the detection of renal and ureteral calculi. However, it is time consuming, expensive, and exposes the patient to i.v. contrast and radiation. To determine the impact of utilizing IVU less for the detection of renal calculi, criteria for ordering IVU in the emergency department (ED) were evaluated, and patients with high probability of positive IVU were identified. Variables included presence of acute flank pain with haematuria, prior history of renal calculus, degree of haematuria, and uncontrolled pain. We reviewed patients presenting with acute flank and abdominal pain with haematuria from May 1995 to May 1996 at a large urban university hospital. Charts were abstracted for prior history, reason for ordering IVU, time in the ED, laboratory results, IVU result, final diagnosis, and disposition. Data was analysed with Student's t-test, Wilcoxon rank-sum and receiver operating characteristic (ROC) analysis. A total of 302 patients were identified, and 185 underwent IVU during the study period. For patients with prior history of renal calculi 82% had positive IVU (sensitivity 51%, specificity 87%). For patients with both acute flank pain and haematuria, 92% had a positive IVU (sensitivity 93%, specificity 43%), and 19% of patients with abdominal pain of unclear aetiology with haematuria had a positive IVU. All patients with uncontrolled pain had evidence of high-grade obstruction on IVU. Degree of haematuria was not predictive of a positive IVU from ROC curve derivation. IVU is a useful study in the ED but may be overutilized, leading to lengthy patient stays. The combined objective findings of acute flank pain and haematuria are sensitive, and prior history is specific in identifying patients with renal calculi. Degree of haematuria was not useful in predicting renal calculi. By utilizing the criteria of acute flank pain and haematuria as a decision aid, 66% of all IVUs ordered could have been avoided.


Histochemical demonstration of adrenergic nerves in the stroma of human cornea.
Both fetal and adult human corneas were subjected to a variety of histochemical techniques for the demonstration of catecholamines. The techniques included both sodium-potassium-glyoxylic acid-induced fluorescence (SPG) and formaldehyde-induced fluorescence after freeze-drying (FIF) procedure. In fetal corneas the presence of adrenergic nerve fibers could be demonstrated by both SPG and FIF techniques. In contrast, in adult human corneas the adrenergic nerves could be identified only with the SPG technique, whereas with FIF the identification of specific catecholamine fluoresence was not possible due to extensive collagen autofluorescence.


Individual differences and specificity of prefrontal gamma frequency-tACS on fluid intelligence capabilities.
Emerging evidence suggests that transcranial alternating current stimulation (tACS) is an effective, frequency-specific modulator of endogenous brain oscillations, with the potential to alter cognitive performance. Here, we show that reduction in response latencies to solve complex logic problem indexing fluid intelligence is obtained through 40 Hz-tACS (gamma band) applied to the prefrontal cortex. This improvement in human performance depends on individual ability, with slower performers at baseline receiving greater benefits. The effect could have not being explained by regression to the mean, and showed task and frequency specificity: it was not observed for trials not involving logical reasoning, as well as with the application of low frequency 5 Hz-tACS (theta band) or non-periodic high frequency random noise stimulation (101-640 Hz). Moreover, performance in a spatial working memory task was not affected by brain stimulation, excluding possible effects on fluid intelligence enhancement through an increase in memory performance. We suggest that such high-level cognitive functions are dissociable by frequency-specific neuromodulatory effects, possibly related to entrainment of specific brain rhythms. We conclude that individual differences in cognitive abilities, due to acquired or developmental origins, could be reduced during frequency-specific tACS, a finding that should be taken into account for future individual cognitive rehabilitation studies.


Relief of sleep apnoea after treatment of acromegaly: report of three cases and review of the literature.
Sleep apnoea syndrome (SAS) is common in acromegalic patients. Occasionally, the relief of apnoeas after treatment of the acromegaly has been documented. We report the cases of three patients with acromegaly and severe obstructive sleep apnoea, who demonstrated a manifest improvement (respiratory disturbance index (RDI) <20) after treatment with octreotide, indicating that this drug may be effective in this disturbance. In one case, SAS disappeared although the growth hormone level was not fully normalized. This raises the intriguing hypothesis that octreotide has an effect on respiratory control or on the upper airway, that is not directly related to its action on production of growth hormone.


Receptors of peptides as therapeutic targets in epilepsy research.
Neuropeptides are signaling molecules participating in the modulation of synaptic transmission. Neuropeptides are stored in dense core synaptic vesicles, the release of which requires profound excitation. Only in the extracellular space, neuropeptides act on G-protein coupled receptors to exert a relatively slow action both pre- and postsynaptically. Consequently, neuropeptide modulators are ideal candidates to influence epileptic tissue overexcited during seizures. Indeed, a number of neuropeptides have receptors implicated in epilepsy and many of them are considered to participate in endogenous neuroprotective actions. Neuropeptide receptors, present in the hippocampus, the most frequent focus of seizures in temporal lobe epilepsy, received the largest attention as potential anti-epileptic targets. Receptors of hippocampal neuropeptides, somatostatin, neuropeptide Y, galanin, dynorphin, enkephalin, substance P, cholecystokinin, vasoactive intestinal polypeptide, and receptors of some neuropeptides, which are also hormones such as ghrelin, angiotensins, corticotropin- releasing hormone, adrenocorticotropin, thyrotropin-releasing hormone, oxytocin and vasopressin involved in epilepsy are discussed in the review article. Activation and inhibition of receptors by oral application of peptides as drugs is typically not efficient because of low bioavailability: rapid degradation and insufficient penetration of peptides through the blood-brain barrier. Recent progress in the development of non-peptide agonists and antagonists of neuropeptide receptors as well as gene therapeutic approaches leading to the local production of agonists and antagonists within the central nervous system will also be discussed.


Applicability of cholesterol-lowering primary prevention trials to a general population: the framingham heart study.
Four large trials have shown cholesterol-reduction therapy to be effective for primary prevention of coronary heart disease (CHD). To determine the generalizability of these trials to a community-based sample, we compared the total cholesterol and high-density lipoprotein cholesterol (HDL-C) distributions of patients in the 4 trials with those of Framingham Heart Study subjects. Lipid profiles that have not been studied were identified. Twelve-year rates of incident CHD were compared between subjects who met eligibility criteria and those who did not. The Framingham sample included 2498 men and 2870 women aged 30 to 74 years. Among Framingham men, 23.4% to 42.0% met eligibility criteria for each of the 4 trials based on their lipid levels; 60.2% met eligibility criteria for at least 1 trial. For the 1 trial that included women, 20.2% of Framingham women met eligibility criteria. In general, subjects with desirable total cholesterol levels and lower HDL-C levels and subjects with average total cholesterol levels and average to higher HDL-C levels have not been included in these trials. Among subjects who developed incident CHD during follow-up, 25.1% of men and 66.2% of women would not have been eligible for any trial. Most ineligible subjects who developed CHD had isolated hypertriglyceridemia (>2.25 mmol/L [>200 mg/dL]). In our sample, 40% of men and 80% of women had lipid profiles that have not been studied in large trials to date. We observed a large number of CHD events in "ineligible" subjects in whom hypertriglyceridemia was common. Further studies are needed to define the role of lipid-lowering therapy vs other strategies for primary prevention in the general population.


A role for vitamin A in the formation of ocular lipofuscin.
Lipofuscin granules in the retinal pigment epithelium are lipid protein aggregates which are thought to represent the lifelong accumulation of the non-degradable end products from the phagocytosis of photoreceptor outer segments. Given the increasing evidence for a key role for vitamin A in the formation of ocular lipofuscin, the fluorophores generated by reacting vitamin A with lipid were assessed. Reaction mixtures consisting of vitamin A (retinol) or its aldehyde (retinal) and (a) isolated rod outer segments, (b) the lipid extract of rod outer segments, (c) protein, or (d) liposomes were incubated at either pH 4.5 or 7.0 for up to 42 days. The fluorescence characteristics and mobility of the chloroform soluble fluorophores generated were compared with those extracted from purified human lipofuscin. Finally, the effect of lysosomal degradation on fluorophores generated in the above mixtures was assessed. Major spectral changes were observed when ROS or liposomes were incubated with retinal. These changes were pH dependent and did not occur if retinal was replaced with retinol. A number of the fluorophores generated exhibited similar fluorescence characteristics and chromatographic mobility to those of lipofuscin. Neither the presence of protein nor exposure to lysosomal enzymes had any effect on the spectral profile or fluorophore mobility of the fluorophores generated. These results suggest that some of the chloroform soluble fluorophores of lipofuscin are formed as a direct reaction product of retinal and lipid.


Geometric morphometrics of the wing in the subgenus Culicoides (Diptera: Ceratopogonidae): from practical implications to evolutionary interpretations.
Biting midges of the genus Culicoides Latreille have been incriminated in transmission of bluetongue. Since 1998, the disease has spread across Europe provoking the largest epidemic ever recorded with important economic loses. Some species of the subgenus Avaritia and Culicoides have been described as candidate vectors involved in these epizootics. Both subgenera contain groups of cryptic species that could differ in their vectorial capacity. For this reason, the correct identification of vector species is considered an essential issue in epidemiological programs. In the current study, the usefulness of wing form in differentiating morphologically similar species of the subgenus Culicoides by means of geometric morphometric techniques is assessed in specimens previously identified through molecular analyses based on cytochrome oxidase subunit I gene sequences. Significant differences between species were detected in the two components of form, i.e., size and shape. Although wing size was affected by temperature, wing shape showed a more stable specific variation, allowing the proper classification of a high percentage of specimens. In addition, the concordance between phylogenies inferred from molecular data and phenetic clusters suggests the existence of a phylogenetic signal in wing shape. These findings enhance the use of this complex phenotypic trait not only to infer genetic relationships among species of the subgenus Culicoides but also as apotentially powerful tool to differentiate cryptic species within the genus.


Economic burden of obesity-related chronic diseases in Mainland China.
The objectives of the present study are: (i) to estimate annual direct medical costs of chronic diseases attributable to overweight and obesity among adults in China and (ii) to predict the medical costs if the epidemic continues developing. Using 2002 National Nutrition and Health Survey (n = 39,834), the prevalence of overweight [24 > or = body mass index (BMI) < 28] and obesity (BMI > or = 28), and population attributable risks (PARs) for hypertension, type 2 diabetes, coronary heart disease and stroke were calculated. The 2003 third National Health Services Survey (n = 143,521) was used to derive direct medical costs including costs for outpatient visits, physician services, inpatient stays, rehabilitation services, nursing fees and medications. The medical costs attributable to overweight and obesity were estimated by multiplying the disease costs by PAR for each disease. The total medical cost attributable to overweight and obesity was estimated at 21.11 billion Yuan (RMB) (approximately $2.74 billion) accounting for 25.5% of the total medical costs for the four chronic diseases, or 3.7% of national total medical costs in 2003. The medical cost associated with overweight and obesity could increase to 37 billion Yuan (RMB) (approximately $4.8 billion), a 75% increase, if the epidemic developed speedily and the ratio of overweight to obesity approached 1.1:1. The high economic burden of overweight and obesity suggests an urgent need to develop effective interventions for controlling the obesity epidemic and consequently the prevention of chronic diseases.


ShRNA-mediated gene silencing of MTA1 influenced on protein expression of ER alpha, MMP-9, CyclinD1 and invasiveness, proliferation in breast cancer cell lines MDA-MB-231 and MCF-7 in vitro.
MTA1(metastasis associated-1) is a tumor metastasis associated candidate gene and overexpression in many human tumors, including breast cancer. In this study, we investigated depressive effect on MTA1 by MTA1-specific short hairpin RNA(shRNA) expression plasmids in human breast cancer cell lines MDA-MB-231 and MCF-7, and effect on protein levels of ER alpha, MMP-9, cyclinD1, and tumor cell invasion, proliferation. ShRNA expression vectors targeting MTA1 was constructed and transfected into human breast cancer cell lines MDA-MB-231 and MCF-7. The transfection efficiency was evaluated by fluorescence microscopy, mRNA levels of MTA1 were detected by reverse transcription-polymerase chain reaction (RT-PCR), protein levels of ER alpha, MMP-9 and cyclinD1 were detected by Western blotting, respectively. Tumor cells invasive ability were evaluated by Boyden chamber assay, the cells proliferation were evaluated using cell growth curve and MTT analysis, the cell cycle analysis was performed using flow cytometry. Down-regulation of MTA1 by RNAi approach led to re-expression of ER alpha in ER-negative breast cancer cell lines MDA-MB-231, and reduced protein levels of MMP-9 and CyclinD1, as well as decreased tumor cell invasion and proliferation, more cells were blocked in G0/G1 stage(P < 0.05). However, after inhibiting mRNA levels of MTA1, protein expression of ER alpha, MMP-9, cyclinD1 and the changes of cancer cells invasiveness, proliferation, cells cycle were no statistical difference in ER-positive human breast cancer cell lines MCF-7 (P > 0.05). ShRNA targeted against MTA1 could specifically mediate the MTA1 gene silencing and consequentially recover the protein expression of ER alpha, resulting in increase sensitivity of antiestrogens, as well as suppress the protein levels of MMP-9 and cyclinD1 in ER-negative human breast cancer cell lines MDA-MB-231. Silencing effect of MTA1 could efficiently inhibit the invasion and proliferation in MDA-MB-231 cells. The shRNA interference targeted against MTA1 may have potential therapeutic utility in human breast cancer.


Parathyroid hormone and the kidney in pseudohypoparathyroidism.
Although a relationship between parathyroid hormone, urinary cyclic AMP and phosphate excretion has been established, the exact molecular sequence which characterizes this cascade is still ambiguous. Consequently, the pathogenesis of inherited forms of hypocalcemia with defective renal responsivity to parathyroid hormone is still conjectural.


The safety of sportive chokes: a cross-sectional survey-based study.
Objectives: Sportive choking or strangling, known as a 'choke' in the combat sports community, is the practice of compressing the jugular veins and carotid arteries to threaten unconsciousness by lowering cerebral perfusion pressure. This is commonly practiced within combat sports and police/military combatives. The safety profile of sportive choking is underrepresented in the literature. The authors sought to explore the safety of sportive chokes. Methods: A convenience sample of visitors to two combat sports internet forums completed an anonymous web-based survey on choking experience and related symptoms. Descriptive statistics were used to describe the obtained data. Bivariate analysis was performed to elaborate on relationships between grappling experience and the number of times choked, between the number of times choked with pre-syncope/syncope, and between the duration of symptoms and the number of times choked with pre-syncope/syncope. Results: Overall, 4421 individuals completed the survey. One hundred and fourteen were excluded, leaving 4307 analyzed respondents. Ninety-four percent were male, 89.2% were ages 18-44 years. Seventy-nine percent had >1 year of grappling experience and 30% had >5 years. Of the 4307, 1443 (33.5%) reported being choked >500 times, 3257 (75.7%) have been choked to near-syncope, and 1198 (27.8%) have been choked unconscious. Two of the 4307 (0.05%) reported ongoing symptoms from chokes. Of the respondents, 94.3% felt applying a choke would be a safe and effective way to control a street fight; 83.6% felt that vascular neck restraint, the police combative equivalent of sportive choking, would be appropriate as an alternative escalation of force option. Conclusion: Based on a convenience sample of 4307 respondents' self-reported data, sportive choking appears to be safe. Only 0.05% experienced ongoing symptoms, which were likely not related to brain ischemia.


Comparative Assessment of Physiological Responses to Emotional Elicitation by Auditory and Visual Stimuli.
The study of emotions through the analysis of the induced physiological responses gained increasing interest in the past decades. Emotion-related studies usually employ films or video clips, but these stimuli do not give the possibility to properly separate and assess the emotional content provided by sight or hearing in terms of physiological responses. In this study we have devised an experimental protocol to elicit emotions by using, separately and jointly, pictures and sounds from the widely used International Affective Pictures System and International Affective Digital Sounds databases. We processed galvanic skin response, electrocardiogram, blood volume pulse, pupillary signal and electroencephalogram from 21 subjects to extract both autonomic and central nervous system indices to assess physiological responses in relation to three types of stimulation: auditory, visual, and auditory/visual. Results show a higher galvanic skin response to sounds compared to images. Electrocardiogram and blood volume pulse show different trends between auditory and visual stimuli. The electroencephalographic signal reveals a greater attention paid by the subjects when listening to sounds compared to watching images. In conclusion, these results suggest that emotional responses increase during auditory stimulation at both central and peripheral levels, demonstrating the importance of sounds for emotion recognition experiments and also opening the possibility toward the extension of auditory stimuli in other fields of psychophysiology. Clinical and Translational Impact Statement- These findings corroborate auditory stimuli's importance in eliciting emotions, supporting their use in studying affective responses, e.g., mood disorder diagnosis, human-machine interaction, and emotional perception in pathology.


Serum fatty acids and progression from dengue fever to dengue haemorrhagic fever/dengue shock syndrome.
PUFA might modulate inflammatory responses involved in the development of severe dengue. We aimed to examine whether serum PUFA concentrations in patients diagnosed with dengue fever (DF) were related to the risk of progression to dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS). A secondary aim was to assess correlations between fatty acids (FA) and inflammatory biomarkers in patients with DF. We conducted a prospective case-control study nested within a cohort of patients who were diagnosed with DF and followed during the acute episode. We compared the distribution of individual FA (% of total FA) at onset of fever between 109 cases who progressed to DHF/DSS and 235 DF non-progressing controls using unconditional logistic regression. We estimated correlations between baseline FA and cytokine concentrations and compared FA concentrations between the acute episode and >1 year post-convalescence in a subgroup. DHA was positively related to progression to DHF/DSS (multivariable adjusted OR (AOR) for DHA in quintile 5 v. 1=5·34, 95 % CI 2·03, 14·1; P trend=0·007). Dihomo-γ-linolenic acid (DGLA) was inversely associated with progression (AOR for quintile 5 v. 1=0·30, 95 % CI 0·13, 0·69; P trend=0·007). Pentadecanoic acid concentrations were inversely related to DHF/DSS. Correlations of PUFA with cytokines at baseline were low. PUFA were lower during the acute episode than in a disease-free period. In conclusion, serum DHA in patients with DF predicts higher odds of progression to DHF/DSS whereas DGLA and pentadecanoic acid predict lower odds.


The human tap nuclear RNA export factor contains a novel transportin-dependent nuclear localization signal that lacks nuclear export signal function.
The human Tap protein mediates the sequence-specific nuclear export of RNAs containing the constitutive transport element and is likely also critical for general mRNA export. Here, we demonstrate that a previously defined arginine-rich nuclear localization signal (NLS) present in Tap acts exclusively via the transportin import factor. Previously, transportin has been shown to mediate the nuclear import of several heterogeneous nuclear ribonucleoproteins, including heterogeneous nuclear ribonucleoprotein (hnRNP) A1, by binding to a sequence element termed M9. Although the Tap NLS and the hnRNP A1 M9 element are shown to compete for transportin binding, they show no sequence homology, and the Tap NLS does not conform to the recently defined M9 consensus. The Tap NLS also differs from M9 in that only the latter is able to act as a nuclear export signal. The Tap NLS is therefore the first member of a novel class of transportin-specific NLSs that lack nuclear export signal function.


Dry and wet seasonal variations of the sediment fungal community composition in the semi-arid region of the Dali River, Northwest China.
Microbial communities play an important role in water quality regulation and biogeochemical cycling in freshwater ecosystems. However, there has been a lack of research on the seasonal variation of sediment microorganisms in the sediments of small river basins in typical semi-arid region. In this study, high-throughput DNA sequencing was used to investigate the fungal community and its influencing factors in the sediment of the Dali River in the dry and wet seasons. The results showed that there were obvious seasonal differences in fungal alpha diversity. The diversity and richness of fungi in the dry season were greater than that in the wet season, but the evenness of fungi in the dry season was lower than that in the wet season. In addition, Ascomycota and Basidiomycota were the most important phyla in the Dali River fungal community, but their distributions showed clear seasonal differences. In the dry season, the relative abundance of Ascomycota and Basidiomycota were 12.34-46.42% and 17.59-27.20%, respectively. In the wet season, the relative abundances of these two phyla were 24.33-36.56% and 5.75-12.26%, respectively. PICRUSt2 was used to predict the metabolic function of fungal community in the sediment, and it was found that at the first level, the proportion of biosynthesis in the dry season was higher than that in the wet season. The ecological network structure showed that the fungal community in the wet season was more complex and stable than that in the dry season. The characteristic fungi in the dry season sediment were chytrid fungi in the family Rhizophydiaceae and the order Rhizophydiales, whereas those in the wet season sediment were in the orders Eurotiales and Saccharomycetales. Canonical correspondence analysis (CCA) showed that the physicochemical properties of water and sediment together explained a greater proportion of the dry-season fungal community changes than of the wet-season changes. In the dry season, temperature and ammonia nitrogen in the water were the main factors affecting the change of fungal community, whereas in the wet season, total nitrogen concentration of the water, electrical conductivity, total organic carbon and available phosphorus of the sediment, pH, and temperature were the main factors affecting the changes in fungal community composition. The results of this study enhanced our understanding of microbial communities in semi-arid river ecosystems, and highlight the importance of the management and protection in river ecosystems.


Closed blastocyst vitrification of biopsied embryos: evaluation of 100 consecutive warming cycles.
The aim of this study was to analyse the outcome of closed blastocyst vitrification of embryos biopsied at the cleavage stage. Vitrification of supernumerary blastocysts was performed using the closed CBS-VIT High Security straws. Warming cycles (n = 100) for patients with preimplantation genetic diagnosis (PGD) and/or aneuploidy screening in the fresh cycle were analysed. The outcome parameters were morphological survival and transfer rates after warming, clinical pregnancy rate and implantation rate (with fetal heart beat). Clinical outcome was compared with two control groups of (i) vitrified/warming transfer cycles without embryo biopsy and (ii) fresh Day 5 transfer of biopsied embryos. In total, 131 blastocysts were warmed with a morphological survival of 83.2% (109/131) and a transfer rate of 79.4% (104/131). Day 5 blastocysts survived significantly better (90.4%) than Day 6 blastocysts (70.8%, P < 0.01). No difference in survival rate was observed between early cavitating (89.2%) and full/expanded blastocysts (93.3%). In nine cycles, no blastocyst was available for transfer. The clinical pregnancy rate was 19.2% (15/78) after single-embryo transfer (SET) and 38.5% (5/13) after double-embryo transfer (DET). In SET, the implantation rate for blastocysts frozen on Day 5 was 13.7% (7/51), which was not different from the implantation rate of Day 6 blastocysts (18.5%, 5/27). The overall implantation rate of vitrified PGD biopsied blastocysts (14.4%) was comparable with that of vitrified blastocysts without biopsy (20.4%), but lower than the implantation rate obtained in the fresh PGD cycles (24.4%). Blastocysts on Day 5 and Day 6 of development derived from biopsied embryos can be successfully vitrified using a closed system.


Shi-style cervical manipulations for cervical radiculopathy: A multicenter randomized-controlled clinical trial.
There is a lack of high-quality evidence supporting the use of manipulation therapy for patients with cervical radiculopathy (CR). This study aimed to evaluate the effectiveness of Shi-style cervical manipulations (SCMs) versus mechanical cervical traction (MCT) for CR. This was a randomized, open-label, controlled trial carried out at 5 hospitals in patients with CR for at least 2 weeks and neck pain. The patients received 6 treatments of SCM (n = 179) or MCT (n = 180) over 2 weeks. The primary outcome was participant-rated disability (neck disability index), measured 2 weeks after randomization. The secondary outcomes were participant-rated pain (visual analog scale) and health-related quality of life (36-Item Short Form Health Survey [SF-36]). Assessments were performed before, during, and after (2, 4, 12, and 24 weeks) intervention. After 2 weeks of treatment, the SCM group showed a greater improvement in participant-rated disability compared with the control group (P = .018). The SCM group reported less disability compared with the control group (P < .001) during the 26-week follow-up. The difference was particularly important at 6 months (mean -28.91 ± 16.43, P < .001). Significant improvements in SF-36 were noted in both groups after 2 weeks of treatment, but there were no differences between the 2 groups. SCM could be a better option than MCT for the treatment of CR-related pain and disability.


Dynamic modelling of cold-hardiness in tea buds by imitating past temperature memory.
Most perennial plants memorize cold stress for a certain period and retrieve the memories for cold acclimation and deacclimation, which leads to seasonal changes in cold-hardiness. Therefore, a model for evaluating cold stress memories is required for predicting cold-hardiness and for future frost risk assessments under warming climates. In this study we develop a new dynamic model of cold-hardiness by introducing a function imitating past temperature memory in the processes of cold acclimation and deacclimation. We formulated the past temperature memory for plants using thermal time weighted by a forgetting function, and thereby proposed a dynamic model of cold-hardiness. We used the buds of tea plants (Camellia sinensis) from two cultivars, 'Yabukita' and 'Yutakamidori', to calibrate and validate this model based on 10 years of observed cold-hardiness data. The model captured more than 90 % of the observed variation in cold-hardiness and predicted accurate values for both cultivars, with root mean square errors of ~1.0 °C. The optimized forgetting function indicated that the tea buds memorized both short-term (recent days) and long-term (previous months) temperatures. The memories can drive short-term processes such as increasing/decreasing the content of carbohydrates, proteins and antioxidants in the buds, as well as long-term processes such as determining the bud phenological stage, both of which vary with cold-hardiness. The use of a forgetting function is an effective means of understanding temperature memories in plants and will aid in developing reliable predictions of cold-hardiness for various plant species under global climate warming.


Detection rates of congenital heart disease in Guatemala.
In developing countries, congenital heart disease is often unrecognised, leading to serious morbidity and mortality. Guatemala is one of the few developing countries where expert paediatric cardiac treatment is available and affordable, and therefore early detection could significantly improve outcome. We assessed regional congenital heart disease detection rates in Guatemala, and determined whether they correlated with the regional human development index. We retrospectively reviewed all new cardiac referrals made in 2006 to the Unidad de Cirugia Cardiovascular Pediatrica, the only paediatric cardiac centre in Guatemala. We calculated regional detection rates by comparing the number of congenital heart disease referrals with the expected incidence using the National Ministry of Health birth data. We then compared the regional detection rates with the human development index data published in the United Nations 2006 Development Program Report using Spearman's rank correlation. An estimated 3935 infants with cardiac defects were born in Guatemala in 2006, an expected 1380 (35%) of whom had severe forms. Overall, only 533 children (14%) with cardiac defects were referred. Of these, 62% had simple shunt lesions, 13% had cyanotic lesions, and 10% had left-sided obstructive lesions. Only 11.5% of referred patients were neonates. Regional detection rates, ranged 3.2-34%, correlated with the regional human development index (r = 0.75, p < 0.0001). Current detection of congenital heart disease in Guatemala is low and correlates with the regional human development index. Those detected are older and have less severe forms, suggesting a high mortality rate among Guatemalan neonates with complex cardiac defects.


Optimized, one-step, recovery-enrichment broth for enhanced detection of Listeria monocytogenes in pasteurized milk and hot dogs.
A one-step, recovery-enrichment broth, optimized Penn State University (oPSU) broth, was developed to consistently detect low levels of injured and uninjured Listeria monocytogenes cells in ready-to-eat foods. The oPSU broth contains special selective agents that inhibit growth of background flora without inhibiting recovery of injured Listeria cells. After recovery in the anaerobic section of oPSU broth, Listeria cells migrated to the surface, forming a black zone. This migration separated viable from nonviable cells and the food matrix, thereby reducing inhibitors that prevent detection by molecular methods. The high Listeria-to-background ratio in the black zone resulted in consistent detection of low levels of L. monocytogenes in pasteurized foods by both cultural and molecular methods, and greatly reduced both false-negative and false-positive results. oPSU broth does not require transfer to a secondary enrichment broth, making it less laborious and less subject to external contamination than 2-step enrichment protocols. Addition of 150mM D-serine prevented germination of Bacillus spores, but not the growth of vegetative cells. Replacement of D-serine with 12 mg/L acriflavin inhibited growth of vegetative cells of Bacillus spp. without inhibiting recovery of injured Listeria cells. oPSU broth may allow consistent detection of low levels of injured and uninjured cells of L. monocytogenes in pasteurized foods containing various background microflora.


[Lymphocytapheresis and lymphosorption as the methods of pathogenetic therapy of nonspecific ulcerative colitis].
The article presents an analysis of results of treatment of 41 patients with different forms of nonspecific ulcer colitis, in 18 of them drainage of thoracic lymphatic duct was used with the following lymphocytapheresis with the help of a home blood fractionator RK-0.5, SKN activated charcoal sorbents and carbohydrate fiber absorbent, lymphosorption and lymph reinfusion. The method of treatment is highly effective for critical and mild forms of ulcer colitis in patients with total and subtotal injury of the large intestine. The results were followed up during 12-30 months and demonstrated stable remission of the disease.


Physiological Signal Monitoring Bed for Infants Based on Load-Cell Sensors.
Ballistocardiographs (BCGs), which record the mechanical activity of the heart, have been a subject of interest for several years because of their advantages in providing unobtrusive physiological measurements. BCGs could also be useful for monitoring the biological signals of infants without the need for physical confinement. In this study, we describe a physiological signal monitoring bed based on load cells and assess an algorithm to extract the heart rate and breathing rate from the measured load-cell signals. Four infants participated in a total of 13 experiments. As a reference signal, electrocardiogram and respiration signals were simultaneously measured using a commercial device. The proposed automatic algorithm then selected the optimal sensor from which to estimate the heartbeat and respiration information. The results from the load-cell sensor signals were compared with those of the reference signals, and the heartbeat and respiration information were found to have average performance errors of 2.55% and 2.66%, respectively. The experimental results verify the positive feasibility of BCG-based measurements in infants.


Improving on nature's shortcomings: evolving a lipase for increased lipolytic activity, expression and thermostability.
An enzyme must be soluble, stable, active and easy to produce to be useful in industrial applications. Not all enzymes possess these attributes. We set out to determine how many changes are required to convert an enzyme with poor properties into one that has useful properties. Lipase Lip3 from Drosophila melanogaster had been previously optimised for expression in Escherichia coli. The expression levels were good, but Lip3 was mainly insoluble with poor activity. Directed evolution was used to identify variants with enhanced activity along with improved solubility. Five variants and the wild-type (wt) enzyme were purified and characterised. The yield of the wt enzyme was just 2.2 mg/L of culture, while a variant, produced under the same conditions, gave 351 mg. The improvement of activity of the best variant was 200 times higher than that of the wt when the crude lysates were analysed using pNP-C8, but with purified protein, the improvement observed was 1.5 times higher. This means that most of the increase of activity is due to increase in solubility and stability. All the purified variants showed increased thermal stability compared with the wt enzyme that had a T1/2 of 37°C, while the mutant with P291L of 42.2°C and the mutant R7_47D with five mutations had a value of 52.9°C, corresponding to an improvement of 16°C. The improved variants had between five and nine changes compared with the wt enzyme. There were four changes that were found in all 30 final round variants for which sequences were obtained; three of these changes were found in the substrate-binding domain.


Solvent and temperature induced switching between structural isomers of Rh(I) phosphinoalkyl thioether (PS) complexes.
To develop functional systems based on the weak-link approach (WLA), it is important to understand how solvent and ligand binding strength alter the coordination geometry of complexes formed from this method. A series of phosphinoalkyl thioether (PS) hemilabile ligands with varying electron donating abilities were synthesized and incorporated into homoligated Rh(I)(PS)2Cl complexes to help understand the effects of solvent and ligand binding strength on the preferred coordination modes. The switching between closed and semiopen structural isomers of these Rh(I)(PS)2Cl complexes was studied by variable temperature 31P NMR spectroscopy in different solvent mixtures of CH2Cl2 and tetrahydrofuran (THF) to obtain thermodynamic parameters (DeltaG(o), DeltaH(o), TDeltaS(o), and K(eq)). The isomers differ in the position of the chloride counterion. In the closed isomer, the Cl- anion occupies the outer coordination sphere, while in the semiopen isomer, the Cl- has moved inner sphere and displaced one of the Rh-S bonds. The closed isomer is favored in CH2Cl2 and the semiopen isomer is favored in THF. The preference for either isomer at equilibrium depends on the solvent polarity, based upon the E(T)(N) solvent polarity scale, as was determined from 15 different solvents, with more polar solvents favoring the closed isomer. The isomer preference also depends on the electron donating ability of the group attached to the sulfur of the PS ligand, with electron donating groups favoring the closed isomers and electron withdrawing groups favoring the semiopen isomers. The formation of the semiopen isomer from the closed isomer is entropically favored but enthalpically disfavored under all conditions studied. Elucidation of the principles and environments that determine the equilibrium between the two isomers will aid in the design of functional complexes prepared by the WLA.


Clinical audit of the use of fresh-frozen plasma and platelets in a tertiary teaching hospital and the impact of a new transfusion request form.
To carry out an audit of the appropriateness of fresh-frozen plasma (FFP) and platelets (Plt) transfusion with reference to the Australian National Health and Medical Research Council/Australian Society of Blood Transfusion Clinical Practice Guidelines, and to assess the impact of a self-educating transfusion request form. A prospective review of the clinical indications and laboratory data in all transfusion episodes of FFP and Plt occurring in a tertiary teaching hospital in South Australia in two 2-month periods of the years 2002 and 2003. Reversal of warfarin has emerged as the major indication to transfuse FFP (34%). More than 72% FFP and 88% Plt were prescribed in an appropriate manner, and the majority were monitored adequately. The transfusion request form further improved the appropriate uses and was met with a satisfactory compliance. Further grounds for improvement are in FFP usage by the haematology unit and in cardiac bypass surgery, and Plt by surgical units. Clinical transfusion audit helps to identify current pattern of usage and areas of improvement. A self-educating transfusion specific request form is also beneficial.


Implicit Theories of Peer Relationships.
This research investigated the role of children's implicit theories of peer relationships in their psychological, emotional, and behavioral adjustment. Participants included 206 children (110 girls; 96 boys; M age = 10.13 years, SD = 1.16) who reported on their implicit theories of peer relationships, social goal orientation, need for approval, depressive and aggressive symptoms, and exposure to peer victimization. Parents also provided reports on aggressive symptoms. Results confirmed that holding an entity theory of peer relationships was associated with a greater tendency to endorse performance-oriented social goals and to evaluate oneself negatively in the face of peer disapproval. Moreover, entity theorists were more likely than incremental theorists to demonstrate depressive and aggressive symptoms when victimized. These findings contribute to social-cognitive theories of motivation and personality, and have practical implications for children exposed to peer victimization and associated difficulties.


The Relation Between Gender Egalitarian Values and Gender Differences in Academic Achievement.
Gender differences in achievement exhibit variation between domains and between countries. Much prior research has examined whether this variation could be due to variation in gender equality in opportunities, with mixed results. Here we focus instead on the role of a society's values about gender equality, which may have a more pervasive influence. We pooled all available country measures on adolescent boys' and girls' academic achievement between 2000 and 2015 from the Program for International Student Assessment (PISA) and Trends in International Mathematics and Science Study (TIMSS) assessments of math, science, and reading. We then analyzed the relation between gender differences and country levels of gender egalitarian values, controlling for country levels of living standards and indicators of gender equality in opportunities. Gender egalitarian values came out as the most important predictor. Specifically, more gender egalitarian values were associated with improved performance of boys relative to girls in the same countries. This pattern held in reading, where boys globally perform substantially worse than girls, as well as in math and science where gender differences in performance are small and may favor either boys or girls. Our findings suggest a previously underappreciated role of cultural values in moderating gender gaps in academic achievement.


TACI deficiency leads to alternatively activated macrophage phenotype and susceptibility to Leishmania infection.
The TNF family member, transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), is a key molecule for plasma cell maintenance and is required in infections where protection depends on antibody response. Here, we report that compared with WT mouse, TACI KO Μϕs expressed lower levels of Toll-like receptors (TLRs), CD14, myeloid differentiation primary response protein 88, and adaptor protein Toll/IL-1 receptor domain-containing adapter-inducing IFN-β and responded poorly to TLR agonists. Analysis of Μϕ phenotype revealed that, in the absence of TACI, Μϕs adapt the alternatively activated (M2) phenotype. Steady-state expression levels for M2 markers IL-4Rα, CD206, CCL22, IL-10, Arg1, IL1RN, and FIZZ1 were significantly higher in TACI KO Μϕ than in WT cells. Confirming their M2 phenotype, TACI-KO Mϕs were unable to control Leishmania major infection in vitro, and intradermal inoculation of Leishmania resulted in a more severe manifestation of disease than in the resistant C57BL/6 strain. Transfer of WT Μϕs to TACI KO mice was sufficient to significantly reduce disease severity. TACI is likely to influence Mϕ phenotype by mediating B cell-activating factor belonging to the TNF family (BAFF) and a proliferation inducing ligand (APRIL) signals because both these ligands down-regulated M2 markers in WT but not in TACI-deficient Μϕs. Moreover, treatment of Μϕs with BAFF or APRIL enhanced the clearance of Leishmania from cells only when TACI is expressed. These findings may have implications for understanding the shortcomings of host response in newborns where TACI expression is reduced and in combined variable immunodeficiency patients where TACI signaling is ablated.


Do Sheriff-Coroners Underreport Officer-Involved Homicides?
In the United States, each state sets its own standards for its death investigation system. These may require independent medical examiners and coroners or allow for the sheriff to assume the role of coroner. Motivated by the well-established fact that counts of officer-involved homicides in official data sets grossly undercount the number of these incidents, we examine the possibility that different death investigation systems may lead to different death classification outcomes. To examine the potential differences in officer-involved homicide underreporting by presence of sheriff-coroner and violent death type (gunshot, intentional use of force, pursuit, or other vehicle accident), we compare ratios of incidents from both the Federal Bureau of Investigation's Supplementary Homicide Reports and the restricted Multiple-Cause of Death files from the National Vital Statistics System to the Fatal Encounters data across coroner contexts in California between 2000 and 2018; we quantify differences descriptively and examine bivariate tests of means. We find significantly greater underreporting of officer-involved deaths in sheriff-coroner counties in both official data sets for all incidents compared with non-sheriff-coroner counties, independently of the period considered. These underreporting differences in the National Vital Statistics System are robust to restricting to gunshot and intentional use of force deaths, the type of incident expected to be less prone to misclassification in that data set. Officer-involved death underreporting in sheriff-coroner counties necessitates further scrutiny. Disparities in officer-involved death reporting suggest political pressure may play a role in classifying deaths.


Multi-Country analysis of palm oil consumption and cardiovascular disease mortality for countries at different stages of economic development: 1980-1997.
Cardiovascular diseases represent an increasing share of the global disease burden. There is concern that increased consumption of palm oil could exacerbate mortality from ischemic heart disease (IHD) and stroke, particularly in developing countries where it represents a major nutritional source of saturated fat. The study analyzed country-level data from 1980-1997 derived from the World Health Organization's Mortality Database, U.S. Department of Agriculture international estimates, and the World Bank (234 annual observations; 23 countries). Outcomes included mortality from IHD and stroke for adults aged 50 and older. Predictors included per-capita consumption of palm oil and cigarettes and per-capita Gross Domestic Product as well as time trends and an interaction between palm oil consumption and country economic development level. Analyses examined changes in country-level outcomes over time employing linear panel regressions with country-level fixed effects, population weighting, and robust standard errors clustered by country. Sensitivity analyses included further adjustment for other major dietary sources of saturated fat. In developing countries, for every additional kilogram of palm oil consumed per-capita annually, IHD mortality rates increased by 68 deaths per 100,000 (95% CI [21-115]), whereas, in similar settings, stroke mortality rates increased by 19 deaths per 100,000 (95% CI [-12-49]) but were not significant. For historically high-income countries, changes in IHD and stroke mortality rates from palm oil consumption were smaller (IHD: 17 deaths per 100,000 (95% CI [5.3-29]); stroke: 5.1 deaths per 100,000 (95% CI [-1.2-11.0])). Inclusion of other major saturated fat sources including beef, pork, chicken, coconut oil, milk cheese, and butter did not substantially change the differentially higher relationship between palm oil and IHD mortality in developing countries. Increased palm oil consumption is related to higher IHD mortality rates in developing countries. Palm oil consumption represents a saturated fat source relevant for policies aimed at reducing cardiovascular disease burdens.


Randomized phase II trial of a toll-like receptor 9 agonist oligodeoxynucleotide, PF-3512676, in combination with first-line taxane plus platinum chemotherapy for advanced-stage non-small-cell lung cancer.
This study assessed the efficacy of the combination of standard taxane plus platinum chemotherapy with the synthetic Toll-like receptor 9-activating oligodeoxynucleotide PF-3512676 in patients with non-small-cell lung cancer (NSCLC). Chemotherapy-naive patients with stage IIIB to IV NSCLC were randomly assigned (one to two ratio) to receive four to six cycles of taxane/platinum chemotherapy alone or with 0.2 mg/kg of subcutaneous PF-3512676 on days 8 and 15 of each 3-week cycle. The primary end point was objective response rate (ORR). Baseline demographics were similar between treatment arms, although significantly more patients in the PF-3512676 arm had stage IV disease (85% compared with 62% in the chemotherapy-alone arm). The modified intent-to-treat analysis (n = 111) demonstrated a 38% ORR (confirmed and unconfirmed) in the PF-3512676 arm (n = 74) and 19% in the chemotherapy-alone arm (n = 37) by investigator evaluation. Blinded, independent radiologic review for 90 patients showed a similar trend in confirmed response rate (19% and 11%, respectively). Median survival was 12.3 months in the PF-3512676 arm and 6.8 months in the chemotherapy-alone arm, and 1-year survival was 50% and 33%, respectively. Mild to moderate local injection site reactions and flu-like symptoms were the most common PF-3512676-related adverse events, but grade 3/4 neutropenia, thrombocytopenia, and anemia were all reported more commonly for patients in the PF-3512676 arm. The addition of PF-3512676 to taxane plus platinum chemotherapy for first-line treatment of NSCLC improves objective response and may improve survival. Confirmatory phase III trials are ongoing.


The protective effects of toothpaste against erosion by orange juice: studies in situ and in vitro.
There has been a monumental increase in the consumption of soft drinks, fruit juices and sport drinks in the UK, the US, and many other countries. Previous investigations have demonstrated the erosive nature of these acidic soft drinks. The aim of this investigation was to determine the protective effects of an experimental fluoride-based toothpaste, containing sodium hexametaphosphate, against an erosive challenge on tooth enamel. The erosion of enamel by orange juice compared the experimental toothpaste with a benchmark sodium fluoride paste and negative control, water, in a 15-day in situ model; and the same in an in vitro enamel erosion model. Flat, polished human enamel samples with a surface profile of +/-0.1mum, were exposed to the three regimens in the single blind, crossover clinical study mimicked in vitro. Depths of the resulting eroded areas were measured using a profilometer. There was significantly more erosive damage on the specimens exposed to the benchmark paste and water compared to the test paste in both the in situ and in vitro studies. The data provide further support for tooth brushing before meals. Results of this study further suggest the sodium hexametaphosphate containing paste could be used to provide significant erosion protection in susceptible individuals over that provided by conventional fluoride products.


Fabrication of embedded microball lens in PMMA with high repetition rate femtosecond fiber laser.
Embedded microball lenses with superior optical properties function as convex microball lens (VMBL) and concave microball lens (CMBL) were fabricated inside a PMMA substrate with a high repetition rate femtosecond fiber laser. The VMBL was created by femtosecond laser-induced refractive index change, while the CMBL was fabricated due to the heat accumulation effect of the successive laser pulses irradiation at a high repetition rate. The processing window for both types of the lenses was studied and optimized, and the optical properties were also tested by imaging a remote object with an inverted microscope. In order to obtain the microball lenses with adjustable focal lengths and suppressed optical aberration, a shape control method was thus proposed and examined with experiments and ZEMAX® simulations. Applying the optimized fabrication conditions, two types of the embedded microball lenses arrays were fabricated and then tested with imaging experiments. This technology allows the direct fabrication of microlens inside transparent bulk polymer material which has great application potential in multi-function integrated microfluidic devices.


Gyroscopic stabilization minimizes drag on Ruellia ciliatiflora seeds.
Fruits of Ruellia ciliatiflora (Acanthaceae) explosively launch small (2.5 mm diameter × 0.46 mm thick), disc-shaped seeds at velocities over 15 m s-1, reaching distances of up to 7 m. Through high-speed video analysis, we observe that seeds fly with extraordinary backspin of up to 1660 Hz. By modelling the seeds as spinning discs, we show that flying with backspin is stable against gyroscopic precession. This stable backspin orientation minimizes the frontal area during flight, decreasing drag force on the seeds and thus increasing dispersal distance. From high-speed video of the seeds' flight, we experimentally determine drag forces that are 40% less than those calculated for a sphere of the same volume and density. This reduces the energy costs for seed dispersal by up to a factor of five.


Statins improve NASH via inhibition of RhoA and Ras.
Nonalcoholic steatohepatitis (NASH), especially as part of the metabolic syndrome (MS), is an increasing burden in Western countries. Statins are already used in MS and seem to be beneficial in liver diseases. The aim of this study was to investigate the molecular mechanisms underlying pleiotropic effects on small GTPases of statins in NASH. NASH within MS was induced in 12-wk-old apoE-/- mice after 7 wk of Western diet (NASH mice). Small GTPases were inhibited by activated simvastatin (SMV), NSC23766 (NSC), or Clostridium sordellii lethal toxin (LT) by using subcutaneous osmotic minipumps. Hepatic steatosis, inflammation, and fibrosis were assessed by histology, Western blot, and RT-PCR measurements of cholesterol and hydroxyproline content. SMV treatment significantly decreased hepatic inflammation and fibrosis, but had no significant effect on steatosis and hepatic cholesterol content in NASH. SMV blunted fibrosis due to inhibition of both RhoA/Rho kinase and Ras/ERK pathways. Interestingly, inhibition of RAC1 and Ras (by LT) failed to decrease fibrosis to the same extent. Inhibition of RAC1 (by NSC) showed no significant effect at all. Inhibition of RhoA and Ras downstream signaling by statins is responsible for the beneficial hepatic effects in NASH.


Citric acid assisted phytoextraction of nickle from soil helps to tolerate oxidative stress and expression profile of NRAMP genes in sunflower at different growth stages.
Soil polluted with Nickel (Ni) adversely affects sunflower growth resulting in reduced yield. Counterbalancing Ni toxicity requires complex molecular, biochemical, and physiological mechanisms at the cellular, tissue, and whole plant levels, which might improve crop productivity. One of the primary adaptations to tolerate Ni toxicity is the enhanced production of antioxidant enzymes and the elevated expression of Ni responsive genes. In this study, biochemical parameters, production of ROS, antioxidants regulation, and expression of NRAMP metal transporter genes were studied under Ni stress in sunflower. There were four soil Ni treatments (0, 50, 100, and 200 mg kg-1 soil), while citric acid (CA, 5 mM kg-1 soil) was applied on the 28th and 58th days of plant growth. The samples for all analyses were obtained on the 30th and 60th day of plant growth, respectively. The results indicated that the concentrations of Ni in roots and shoots were increased with increasing concentrations of Ni at both time intervals. Proline contents, ascorbic acid, protein, and total phenolics were reduced under Ni-stress, but with the application of CA, improvement was witnessed in their contents. The levels of malondialdehyde and hydrogen peroxide were enhanced with the increasing concentration of Ni, and after applying CA, they were reduced. The contents of antioxidants, i.e., catalase, peroxidase, superoxide dismutase, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase, were increased at 50 ppm Ni concentration and decreased at higher concentrations of Ni. The application of CA significantly improved antioxidants at all concentrations of Ni. The enhanced expression of NRAMP1 (4, 51 and 81 folds) and NRAMP3 (1.05, 4 and 6 folds) was found at 50, 100 and 200ppm Ni-stress, respectively in 30 days old plants and the same pattern of expression was recorded in 60 days old plants. CA further enhanced the expression at both developmental stages. In conclusion, CA enhances Ni phytoextraction efficiency as well as protect plant against oxidative stress caused by Ni in sunflower.


Conformational sampling, catalysis, and evolution of the bacterial phosphotriesterase.
To efficiently catalyze a chemical reaction, enzymes are required to maintain fast rates for formation of the Michaelis complex, the chemical reaction and product release. These distinct demands could be satisfied via fluctuation between different conformational substates (CSs) with unique configurations and catalytic properties. However, there is debate as to how these rapid conformational changes, or dynamics, exactly affect catalysis. As a model system, we have studied bacterial phosphotriesterase (PTE), which catalyzes the hydrolysis of the pesticide paraoxon at rates limited by a physical barrier-either substrate diffusion or conformational change. The mechanism of paraoxon hydrolysis is understood in detail and is based on a single, dominant, enzyme conformation. However, the other aspects of substrate turnover (substrate binding and product release), although possibly rate-limiting, have received relatively little attention. This work identifies "open" and "closed" CSs in PTE and dominant structural transition in the enzyme that links them. The closed state is optimally preorganized for paraoxon hydrolysis, but seems to block access to/from the active site. In contrast, the open CS enables access to the active site but is poorly organized for hydrolysis. Analysis of the structural and kinetic effects of mutations distant from the active site suggests that remote mutations affect the turnover rate by altering the conformational landscape.


Selection and enrichment of antigen-specific T lymphocyte populations.
Antigen-specific T lymphocytes bind in vitro to cells carrying alloantigens or to macrophages bearing antigen to which the lymphocytes were sensitized. This phenomenon is the basis of present procedures for the isolation and enrichment of antigen-specific T lymphocytes. The antigen-mediated adherence permits the removal of T cells with other specificities, which do not bind to the antigen-containing monolayer. The subsequent activation and multiplication of the antigen-bound cells further increases the proportion of the antigen-specific lymphocytes in culture. The selected cells, which are derived from a small fraction of the initial lymphocyte population, proliferate until they comprise up to 30% of the original total cell number. Another factor favoring enrichment is that unactivated cells do not survive well in culture whereas antigen-specific activated cells maintain their viability. Indeed, the selected cells can be maintained for a few weeks in culture. The activity of the selected lymphocytes is elevated toward the antigen used for enrichment, but lowered, or absent, toward other antigens. The progeny of the antigen-adherent lymphocytes are thus seen to be enriched for one antigen-specific subpopulation, and deprived of lymphocytes committed to other antigens. Cells obtained by this procedure should prove to be very useful for the functional analysis of T lymphocytes.


9-16 Hz oscillation precedes secondary generalization of seizures in the rat tetanus toxin model of epilepsy.
Unilateral intrahippocampal injection of tetanus toxin results in a chronic syndrome of intermittent epileptic seizures. During some of these seizures, rats develop a stereotypic, pathological motor behavior that indicates secondary generalization of epileptic activity. We report that secondary generalization was preceded by a 9-16 Hz oscillation of field potentials which was synchronized between the right and left dorsal hippocampi. The oscillation was associated with increased synchrony of population spike firing in right and left CA1 subregions which form the major output of the hippocampi. Cutting the ventral commissure abolished synchrony across the hippocampi and reduced the probability that the 9-16 Hz activity would be followed by secondary generalization. We concluded that a bilaterally synchronous 9-16 Hz hippocampal oscillation played a role in the secondary generalization of focal seizures in this chronic model of limbic epilepsy.


Inkjet printing of layer-by-layer assembled poly(lactide) stereocomplex with encapsulated proteins.
Inkjet printing, a technique that precisely deposits liquid droplets in picoliter-volume ranges on a substrate, has received increased attention for its novelty and ability to produce functional materials. This technology is considered one of the most promising methods for the controlled deposition of different polymers. In our previous study, a poly(lactide) (PLA) stereocomplex was fabricated using inkjet printing on a substrate. The stereocomplex was formed by the layer-by-layer (LbL) stepwise deposition of poly(l-lactide) (PLLA) and poly(d-lactide) (PDLA). Multiple inkjet passes could conclusively improve the PLAs crystal structure with solvent evaporation (solidification) and dissolution of PLA. We suggested that this technique may also be applicable for fabricating polymer composites with drugs, such as peptides, proteins, and nanoparticles, which is incompatible with the PLA. Here, we report the utilization of this technique to create a PLA stereocomplex with drugs as a drug carrier/reservoir. The three components of PLLA, PDLA, and model drugs (an 8-mer peptide, ovalbumin, and protein-encapsulating nanoparticles) were alternately overprinted onto the substrate without an intermediate rinsing step. Inkjet printing was used successfully to form PLA stereocomplex composites with drugs by the LbL deposition of polymers and functioned as drug carriers/reservoirs. The sustained release of the drugs was observed from the PLLA/PDLA/drug composites. By varying the crystalline structure of PLAs-drug composites, the release kinetics of drugs could be altered and controlled efficiently. Moreover, a high drug loading content (wt %) of PLA stereocomplex composites was achieved up to 100 wt % loading, and the composites with 50 wt % of drug loading content were available for sustained-release formulation. This fabrication technique would provide a platform for creating protein/vaccine/gene delivery carriers with the desired release profiles by controlling the microphase-separated structure and drug distribution within the composites.


Profiling of miRNAs in porcine germ cells during spermatogenesis.
Spermatogenesis includes mitosis of spermatogonia, meiosis of pachytene spermatocytes and spermiogenesis of round spermatids. MiRNAs as a ~22 nt small noncoding RNA are involved in regulating spermatogenesis at post-transcriptional level. However, the dynamic miRNAs expression in the developmental porcine male germ cells remains largely undefined. In this study, we purified porcine spermatogonia, pachytene spermatocytes and round spermatids using a STA-PUT apparatus. A small RNA deep sequencing and analysis were conducted to establish a miRNAs profiling in these male germ cells. We found that 19 miRNAs were differentially expressed between spermatogonia and pachytene spermatocytes, and 74 miRNAs differentially expressed between pachytene spermatocytes and round spermatids. Furthermore, 91 miRNAs were upregulated, while 108 miRNAs were downregulated in spermatozoa. We demonstrated that ssc-miR-10a-5p, ssc-miR-125b, ssc-let-7f and ssc-miR-186 were highly expressed in spermatogonia, pachytene spermatocytes, round spermatids and spermatozoa respectively. The findings could provide novel insights into roles of miRNAs in regulation of porcine spermatogenesis.


Ab initio study of carbon-chlorine bond cleavage in carbon tetrachloride.
Chlorinated solvents in groundwater are known to undergo reductive dechlorination reactions with Fe(ll)-containing minerals and with corroding metals in permeable-barrier treatment systems. This research investigated the effect of the reaction energy on the reaction pathway for C-Cl bond cleavage in carbon tetrachloride (CCl4). Hartree-Fock, density functional theory, and modified complete basis set ab initio methods were used to study adiabatic electron transfer to aqueous-phase CCl4. The potential energies associated with fragmentation of the carbon tetrachloride anion radical (CCl4-) into a trichloromethyl radical (CCl3) and a chloride ion (Cl-) were explored as a function of the carbon-chlorine bond distance during cleavage. The effect of aqueous solvation was investigated using a continuum conductor-like screening model. Solvation significantly lowered the energies of the reaction products, suggesting that dissociative electron transfer was enhanced by solvation. The potential energy curves in an aqueous medium indicate that reductive cleavage undergoes a change from an inner-sphere to an outer-sphere mechanism as the overall energy change for the reaction is increased. The activation energy for the reaction was found to be a linear function of the overall energy change, and the Marcus-Hush model was used to relate experimentally measured activation energies for CCl4 reduction to overall reaction energies. Experimentally measured activation energies for CCl4 reduction by corroding iron correspond to reaction energies that are insufficiently exergonic for promoting the outer-sphere mechanism. This suggests that the different reaction pathways that have been observed for CCl4 reduction by corroding iron arise from different catalytic interactions with the surface, and not from differences in energy of the transferred electrons.


Silencing of Irf7 pathways in breast cancer cells promotes bone metastasis through immune escape.
Breast cancer metastasis is a key determinant of long-term patient survival. By comparing the transcriptomes of primary and metastatic tumor cells in a mouse model of spontaneous bone metastasis, we found that a substantial number of genes suppressed in bone metastases are targets of the interferon regulatory factor Irf7. Restoration of Irf7 in tumor cells or administration of interferon led to reduced bone metastases and prolonged survival time. In mice deficient in the interferon (IFN) receptor or in natural killer (NK) and CD8(+) T cell responses, metastasis was accelerated, indicating that Irf7-driven suppression of metastasis was reliant on IFN signaling to host immune cells. We confirmed the clinical relevance of these findings in over 800 patients in which high expression of Irf7-regulated genes in primary tumors was associated with prolonged bone metastasis-free survival. This gene signature may identify patients that could benefit from IFN-based therapies. Thus, we have identified an innate immune pathway intrinsic to breast cancer cells, the suppression of which restricts immunosurveillance to enable metastasis.


Sodium-pyrazinoate cotransport in rabbit renal brush border membrane vesicles.
Pyrazinoate (PZA) is an organic anion actively reabsorbed and secreted in the mammalian kidney. In experiments with rabbit renal brush border membrane vesicles, we characterized a sodium-PZA cotransport mechanism that could be involved in reabsorption. An inwardly directed sodium gradient stimulated the influx of PZA. The sodium-dependent transport was electroneutral, suggesting a 1:1 stoichiometry. The kinetic constants for sodium-PZA cotransport were measured under initial linear flux and zero trans conditions for both sodium and PZA. The apparent Km for sodium was about 60 mM. At 90 mM sodium the apparent Km for PZA was about 1.1 mM; increasing the sodium concentration augmented the apparent affinity for PZA. Cis inhibition of sodium-dependent PZA uptake was observed by the addition of nicotinate, lactate, probenecid, succinate, beta-hydroxybutyrate, and salicylate. Urate had no effect. [14C]PZA uptake was trans stimulated by PZA itself, lactate, and nicotinate. PZA shares a transport system(s) involved in the proximal tubular reabsorption of these two anions.


Increased hepatic expression of dipeptidyl peptidase-4 in non-alcoholic fatty liver disease and its association with insulin resistance and glucose metabolism.
Dipeptidyl peptidase-4 (DPP4) is a serine protease that degrades glucagon-like peptide-1 (GLP-1), an incretin hormone that stimulates insulin secretion from pancreatic β-cells. DPP4 is also involved in the regulation of T cell-mediated inflammatory processes. These properties of DPP4 suggest that it may play a role in the progression of non-alcoholic fatty liver disease (NAFLD). Hepatic DPP4 mRNA expression levels were analyzed by real-time PCR using liver biopsy samples from 17 NAFLD patients and 10 healthy subjects. In NAFLD patients, we also examined correlations between DPP4 expression levels and metabolic factors, including homeostasis model assessment-insulin resistance (HOMA-IR), body mass index (BMI), and serum cholesterol and triglyceride levels. To examine the potential effects of nutritional factors, DPP4 expression levels were analyzed in HepG2 cells subjected to various culture conditions. Hepatic DPP4 mRNA expression was significantly greater in NAFLD patients than in control subjects. DPP4 expression levels were negatively correlated with HOMA-IR and positively correlated with serum cholesterol levels. In HepG2 cells, high glucose significantly enhanced DPP4 expression, whereas insulin, fatty acids and cholesterol did not. Increased hepatic expression of DPP4 in NAFLD may be associated with metabolic factors, including insulin resistance, and may adversely affect glucose metabolism in this liver disease.


Pain-Related Fear-Dissociable Neural Sources of Different Fear Constructs.
Fear of pain demonstrates significant prognostic value regarding the development of persistent musculoskeletal pain and disability. Its assessment often relies on self-report measures of pain-related fear by a variety of questionnaires. However, based either on "fear of movement/(re)injury/kinesiophobia," "fear avoidance beliefs," or "pain anxiety," pain-related fear constructs plausibly differ while it is unclear how specific the questionnaires are in assessing these different constructs. Furthermore, the relationship of pain-related fear to other anxiety measures such as state or trait anxiety remains ambiguous. Advances in neuroimaging such as machine learning on brain activity patterns recorded by functional magnetic resonance imaging might help to dissect commonalities or differences across pain-related fear constructs. We applied a pattern regression approach in 20 human patients with nonspecific chronic low back pain to reveal predictive relationships between fear-related neural pattern information and different pain-related fear questionnaires. More specifically, the applied multiple kernel learning approach allowed the generation of models to predict the questionnaire scores based on a hierarchical ranking of fear-related neural patterns induced by viewing videos of activities potentially harmful for the back. We sought to find evidence for or against overlapping pain-related fear constructs by comparing the questionnaire prediction models according to their predictive abilities and associated neural contributors. By demonstrating evidence of nonoverlapping neural predictors within fear-processing regions, the results underpin the diversity of pain-related fear constructs. This neuroscientific approach might ultimately help to further understand and dissect psychological pain-related fear constructs.


A Primer for Evaluating Clinical Trials.
BACKGROUND: Evidence-based medicine demands the use of information from clinical trials to direct medical care. Knowledge of the principles of trial design and conduct is important to assess the validity of results. METHODS: The authors review the key principles behind clinical study design and conduct, and they summarize important biases and confounding issues. RESULTS: Clear hypotheses, a well-described study population, precise measurements, freedom from bias, and consideration of any interactions are attributes of good clinical trials. CONCLUSIONS: The greatest level of evidence in support of a difference in outcome is associated with randomized, controlled clinical trials, particularly when combined with other randomized trials in a systematic fashion (meta-analysis).