Possible involvement of the activation of voltage-sensitive calcium channels in the ameliorating effects of nefiracetam on scopolamine-induced impairment of performance in a passive avoidance task.
We investigated the effects of various types of calcium channel antagonists on the amelioration by nefiracetam [N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl) acetamide, DM-9384] of scopolamine-induced impairment of performance in a passive avoidance task in mice. The reversal of the scopolamine-induced impairment of performance by nefiracetam showed a bell-shaped plot. Both i.p. and i.c.v. injection of L-type calcium channel antagonists such as nifedipine and flunarizine attenuated the ameliorating effects of nefiracetam, although diltiazem had no effect. Neomycin, an N-type calcium channel antagonist, also attenuated these effects of nefiracetam in a dose-dependent manner. Further, LaCl3 but not NiCl2 showed inhibitory effects on the amelioration by nefiracetam. These results suggest that the activation of high-voltage-activated, but not low-voltage-activated, calcium channels is involved in the ameliorating effects of nefiracetam on scopolamine-induced impairment of performance in a passive avoidance task.


MiR-539-3p impairs osteogenesis by suppressing Wnt interaction with LRP-6 co-receptor and subsequent inhibition of Akap-3 signaling pathway.
X-linked hypophosphatemia (XLH), an inheritable form of rickets is caused due to mutation in Phex gene. Several factors are linked to the disease's aetiology, including non-coding RNA molecules (miRNAs), which are key post-transcriptional regulators of gene expression and play a significant role in osteoblast functions. MicroRNAs sequence analysis showed differentially regulated miRNAs in phex silenced osteoblast cells. In this article, we report miR-539-3p, an unidentified novel miRNA, in the functional regulation of osteoblast. MiR-539-3p overexpression impaired osteoblast differentiation. Target prediction algorithm and experimental confirmation by luciferase 3' UTR reporter assay identified LRP-6 as a direct target of miR-539-3p. Over expression of miR-539-3p in osteoblasts down regulated Wnt/beta catenin signaling components and deteriorated trabecular microarchitecture leading to decreased bone formation in ovariectomized (Ovx) mice. Additionally, biochemical bone resorption markers like CTx and Trap-5b were elevated in serum samples of mimic treated group, while, reverse effect was observed in anti-miR treated animals along with increased bone formation marker P1NP. Moreover, transcriptome analysis with miR-539-3p identified a novel uncharacterized Akap-3 gene in osteoblast cells, knock down of which resulted in downregulation of osteoblast differentiation markers at both transcriptional and translational level. Overall, our study for the first time reported the role of miR-539-3p in osteoblast functions and its downstream Akap-3 signalling in regulation of osteoblastogenesis.


[Application of VBA programming to achieve high quality critical illness score].
To establish a high-quality critical illness scoring program by VBA programming. The acute physiology and chronic health evaluatin II (APACHE II), Logistic organ dysfunction score (LODS) and sequential organ failure assessment (SOFA) were combined into a program with visual basic for applications (VBA) of Microsoft Excel. Taking the case data published in the literature as an example, the program was applied to SOFA score. First, directly copied the patient's data, such as age, body weight, body temperature, pulse, blood pressure, blood routine, blood biochemistry, blood gas analysis, etc. Then, returned to the program, clicked the "paste data" button, and the data would be automatically filled in, and selected the worst option. Then, the usage of norepinephrine, mechanical ventilation, Glasgow coma scale (GCS) score and 24-hour urine volume were input according to the situation. Finally, clicked the "score" button to get SOFA score. Pasted the data directly into the critical illness scoring program and checked it accordingly. The total time from data input to abtaining scoring result was less than 2 minutes; and the repeatability was strong. The application of VBA self-programming can quickly and accurately perform critical illness scoring.


[Crusted scabies (Norwegian scabies) a case report].
Different types of scabies have been described based on their clinical outcome, one of which is the Crusted (Norwegian) type. This is an extreme manifestation of scabies that can be observed mainly among immunosupressed patients. A case ofa 42 year-old homosexual man is described. The patient was diagnosed with HIV, presenting pruritic lesions with a 4 month evolution in trunk and extremities. Lesions included xerosis, decapitated papules, badges with erythema, residual hyperchromic stains, multiple abrasions and ungueal pigmentation in both feet. At the beginning it was treated as apsorasiform dermatitis with steroids and antipruritics without success. Through a biopsy the suspected diagnosis of Crusted (Norwegian) scabies was confirmed. The patient was treated with a dose of oral ivermectin and topical benzyl benzoate and showed remission after two days.


Chronological Development of Cardiovascular Disease in Times of COVID-19: A Retrospective Analysis of Hospitalized Diseases of the Circulatory System and COVID-19 Patients of a German University Hospital.
This study aims at examining the chronological development of hospitalized cardiovascular and COVID-19 patients and comparing the effects on related sub-disciplines and main diagnoses for pre-pandemic (2017-2019) and pandemic (2020-2021) years in the setting of a German university maximum care provider. Data were retrospectively retrieved from the hospital performance controlling system for patient collectives with main diagnosis of diseases of the circulatory system (nCirculatory) and COVID-19 secondary diagnosis (nCOVID-19). The cardiovascular patient collective (nCirculatory = 25,157) depicts a steady state in terms of relative yearly development of patient numbers (+0.4%, 2019-2020, +0.1%, 2020-2021). Chronological assessment points towards monthly decline during lockdowns and phases of high regional incidence of COVID-19 (i.e., 2019-2020: March -10.2%, April -12.4%, December -14.8%). Main diagnoses of congestive heart failure (+16.1% 2019/2020; +19.2% 2019/2021) and acute myocardial infarction show an increase in case numbers over the course of the whole pandemic (+15.4% 2019/2020; +9.4% 2019/2021). The results confirm negative effects on the cardiovascular care situation during the entire pandemic in the setting of a university maximum care provider. A general increase in cardiac disorders and a worrisome turn in case development of acute myocardial infarction emphasize the feared cardiovascular burden of COVID-19.


Poly (ADP-ribose) polymerase inhibitors (PARPi) in ovarian cancer: lessons learned and future directions.
Poly (ADP-ribose) polymerase inhibitors (PARPi) represent a new standard of care in the upfront treatment of advanced epithelial ovarian cancer to the point that the vast majority of patients now receive a PARPi, alone or in combination with the anti-angiogenic bevacizumab, as part of their first-line maintenance therapy. The clinical benefit of PARPi is well established; however, much has changed since their introduction and several relevant questions have been raised and remain unresolved in the post-PARPi era. The decision-making process regarding the most appropriate first-line maintenance therapy could be challenging in clinical practice, especially in the homologous recombination-proficient setting, and several other factors need to be considered apart from the mutational status. Concerns regarding post-PARPi progression treatment have emerged, highlighting an unmet need to define a valid algorithm strategy. PARPi may not only compromise the response to further platinum due to cross-resistance mechanisms but the impact on subsequent non-platinum chemotherapy and surgery also remains unclear. Definitive results on the role of PARPi rechallenge are awaited, especially in the case of oligoprogression managed with locoregional treatment. Moreover, the updated overall survival data from the recurrent setting warrant caution in using PARPi as single agents for unselected patients. Several PARPi combination regimens are emerging for overcoming PARPi resistance and may become our new therapeutic armamentarium. This review discusses a set of clinically relevant issues in the PARPi era and provides a glimpse of future challenges and opportunities in ovarian cancer treatment.


Circadian melatonin concentration rhythm is lost in pregnant women with altered blood pressure rhythm.
We assessed the correlation between the rhythm of melatonin concentration and circadian blood pressure patterns in normal and hypertensive pregnancy. Ambulatory 24-h blood pressure and blood samples every 4 h were monitored in 16 primigravidae who had shown an abnormal circadian blood pressure pattern (eight pre-eclamptic and eight normotensive) in pregnancy and 6-12 months after pregnancy. The circadian rhythm was analyzed by chronobiological measures. Eight normotensive women with maintained blood pressure rhythm served as controls. During pregnancy, melatonin concentration was significantly higher in pre-eclamptic than in normotensive women (pre-eclampsia, 29.4 +/- 1.9 pg/ml, normotensin, altered rhythm, 15.6 +/- 2.1; controls, 22.7 +/- 1.8; p < 0.001). This difference faded after pregnancy, owing to the fall observed in pre-eclampsia (11.8 +/- 3.2 pg/ml, 9.8 +/- 2.1, and 11.1 +/- 2.0, respectively; NS). The rhythm of melatonin concentration was lost in all pregnant women with loss of blood pressure rhythm. After pregnancy, normotensive women showed a reappearance of both melatonin and blood pressure rhythm, whereas pre-eclamptic women showed a reappearance of blood pressure but not melatonin rhythm. The loss of blood pressure rhythm in pregnancy is consistent with the loss of melatonin concentration rhythm. In pre-eclamptic women, the normalization of blood pressure rhythm, while melatonin rhythm remained altered, suggests a temporal or causal priority of circadian concentration of melatonin in the determination of blood pressure trend.


The contribution of anti-prothrombin-antibodies to lupus anticoagulant activity--discrimination between functional and non-functional anti-prothrombin-antibodies.
The presence of lupus anticoagulant (LAC) is strongly correlated with a history of thrombosis in patients with SLE. LAC activity can be caused by anti-prothrombin (FII)- and/or anti-beta2glycoprotein I (beta2GPI)-antibodies. In the present study, the contribution of anti-FII-antibodies to LAC activity was measured in 28 LAC positive plasmas. Plasmas were incubated with prothrombin or BSA, immobilized on CNBr-activated Sepharose, to absorb all anti-FII-antibodies. In 4 out of the 28 plasmas LAC activity was completely dependent on anti-FII-antibodies. In 7 out of the 28 plasmas, anti-FII-antibodies did not contribute to LAC activity. These anti-FII-antibodies can be regarded as non-functional antibodies. In the majority (17/28) of the samples, LAC activity within a single plasma was caused by a combination of antibodies with different specificities. Both dRVVT and KCT showed comparable sensitivity for the detection of functional anti-FII-antibodies. In conclusion, in most samples LAC activity is not caused by anti-FII-antibodies alone but by a combination of different types of antibodies. The presence of LAC activity and anti-FII-antibodies in one plasma does not automatically implicate that these antibodies are responsible for the LAC activity.


Physical Activity Types and Health-Related Quality of Life among Middle-Aged and Elderly Adults: The Rotterdam Study.
Physical activity (PA) is associated with health-related quality of life (HRQL). The specific PA types that provide beneficial effects in an older population remain unclear. We assessed the association of total PA, walking, cycling, domestic work, sports and gardening with HRQL in middle-aged and elderly adults. Cross-sectional study. Rotterdam, the Netherlands. 5,554 participants, with a mean age of 69 years. Total PA was categorized in five groups to evaluate the dose-response effect of PA and specific PA types were categorized in tertiles. HRQL was measured with the EuroQoL 5-dimension. The outcome of every HRQL domain (i.e. mobility, self-care, daily activities, pain and mood) was expressed as having any problems versus not having problems. Logistic and linear regression analyses were used, adjusting for confounders, to examine associations of total PA and PA types with HRQL domains. In both middle-aged (<65 years) and elderly adults (>65 years), we found a dose-response association between total PA and better HRQL (i.e. lower odds of having problems in HRQL domains). In the middle-aged, sports was the only PA type associated with lower odds of having problems with all HRQL domains. In the elderly, all PA types were associated with less problems with HRQL domains, but cycling contributed most to the beneficial effect. Total PA was associated with better HRQL. Sports and cycling were the activity types that contributed most to this association in the middle-aged and elderly, respectively. Since PA levels tend to decline with aging, cycling and sports should be promoted with the aim to improve HRQL.


Renal tubular necrosis due to shock: light and electron-microscope observations.
The renal biopsy findings in a 76 yr-old woman suffering grom anuria due to acute tubular necrosis are described. The glomeruli were normal on light- and electron microscopy. Immunofluorescent studies failed to reveal any fibrin or immunoglobulins in the glomerular capillaries. Extensive focal areas of necrosis were seen in the tubular epithelium often exposing the lumen of the tubule directly to the tubular basement-membrane. In some areas necrotic cells lay adjacent to normal or near normal cells. The proximity of the necrotic tubular epithelium to the oedematous interstitial tissue and the peritubular capillaries, together with the finding of normal glomeruli is compatible with the theory of back diffusion as a mechanism for the oliguria.


Dairy Food Intake and All-Cause, Cardiovascular Disease, and Cancer Mortality: The Golestan Cohort Study.
We investigated the association between dairy product consumption and all-cause, cardiovascular disease (CVD), and cancer mortality in the Golestan Cohort Study, a prospective cohort study launched in January 2004 in Golestan Province, northeastern Iran. A total of 42,403 men and women participated in the study and completed a diet questionnaire at enrollment. Cox proportional hazards models were used to estimate hazard ratios and 95% confidence intervals. We documented 3,291 deaths (1,467 from CVD and 859 from cancer) during 11 years of follow-up (2004-2015). The highest quintile of total dairy product consumption (versus the lowest) was associated with 19% lower all-cause mortality risk (hazard ratio (HR) = 0.81, 95% confidence interval (CI): 0.72, 0.91; Ptrend = 0.006) and 28% lower CVD mortality risk (HR = 0.72, 95% CI: 0.60, 0.86; Ptrend = 0.005). High consumption of low-fat dairy food was associated with lower risk of all-cause (HR = 0.83, 95% CI: 0.73, 0.94; Ptrend = 0.002) and CVD (HR = 0.74, 95% CI: 0.61, 0.89; Ptrend = 0.001) mortality. We noted 11% lower all-cause mortality and 16% lower CVD mortality risk with high yogurt intake. Cheese intake was associated with 16% lower all-cause mortality and 26% lower CVD mortality risk. Higher intake of high-fat dairy food and milk was not associated with all-cause or CVD mortality. Neither intake of individual dairy products nor intake of total dairy products was significantly associated with overall cancer mortality. High consumption of dairy products, especially yogurt and cheese, may reduce the risk of overall and CVD mortality.


Greater Adherence to Recommended Morning Physical Activity is Associated With Greater Total Intervention-Related Physical Activity Changes in Bariatric Surgery Patients.
This study examines whether performance of bout-related physical activity (PA) during morning hours is related to greater overall bout-related PA increases within a preoperative PA intervention for bariatric surgery (BS) patients. Participants with severe obesity (n = 33; mean age = 45.6 ± 9.6 years; BMI = 45.7 ± 7.0 kg/m2) seeking BS were randomized to and completed 6 weeks of preoperative PA counseling (retention = 82.5%). Participants were encouraged to walk daily at a moderate intensity in bouts ≥ 10 minutes during morning hours to overcome time-related obstacles and establish a PA habit. Timing and amount of bout-related moderate-to-vigorous PA (MVPA) was assessed via objective monitor at pre- and postintervention. Greater proportion of bout-related MVPA performed during morning hours (4:00 AM-12:00 PM) at postintervention was associated with larger total increases in bout-related MVPA minutes/day (β = .40, P = .03). At postintervention, a greater proportion of participants whose longest MVPA bouts occurred during morning hours (n = 11) achieved the public health guideline (ie, ≥150 bout-related MVPA minutes/week) versus those whose longest MVPA bouts occurred during nonmorning hours (n = 19; 63.6% vs. 26.3%, P = .04). Intervention-related increases in PA tended to be greatest when PA was performed in the morning. Morning exercise may be a viable strategy for promoting habitual PA in inactive BS patients.


Plotting Kinetic Data To Determine Reaction Order and Specific Rate Constant.
The Pessen method for obtaining from kinetic experiments both order of reaction and rate constant by means of single straight-line graphs has been combined with the concept of unit time to permit handling of data over the range of 0 to 99 percent conversion or reaction.


Signatures of dynamical tunneling in semiclassical quantum dots.
We study transport in large, and strongly open, quantum dots, which might typically be viewed as lying well within the semiclassical regime. The low-temperature magnetoresistance of these structures exhibits regular fluctuations, with just a small number of dominant frequency components, indicative of the presence of dynamical tunneling into regular orbits. Support for these ideas is provided by the results of numerical simulations, which reveal wave function scarring by classically inaccessible orbits, which is found to persist even in the presence of a moderately disordered dot potential. Our results suggest that dynamical tunneling may play a more generic role in transport through mesoscopic structures than has thus far been appreciated.


Annotated checklist of earthworm species described by András Zicsi (Clitellata: Megadrili).
Professor András Zicsi, the renowed taxonomist and ecologist of earthworms, passed away on 22 July, 2015. He had a decisive impact on earthworm taxonomy in the second half of the 20th century, describing 10 new genus group taxa and 243 species / subspecies new to science distributed in 10 families. A complete list of the new taxa described by Prof. Zicsi is presented here together with references to the original descriptions and the valid names.


Effect of ts mutation in gene 43 of bacteriophage T4 on recombination of the bacteriophage.
Certain ts mutations in gene 43 of bacteriophage T4 are known to exhibit mutator or antimutator activities with respect to different rII mutations of this phage. The effect of these mutations on recombination frequencies of double mutant strains of phage T4 with genotype rII ts--homoallelic with respect to the ts trait--was examined. The results implicate the essential role of the genetic background in the investigated process.


Zinc Absorption from Representative Diet in a Chinese Elderly Population Using Stable Isotope Technique.
To determine the dietary zinc absorption in a Chinese elderly population and provide the basic data for the setting of zinc (Zn) recommended nutrient intakes (RNI) for Chinese elderly people. A total of 24 elderly people were recruited for this study and were administered oral doses of 3 mg 67Zn and 1.2 mg dysprosium on the fourth day. The primary macronutrients, energy, and phytic acid in the representative diet were examined based on the Chinese National Standard Methods. Fecal samples were collected during the experimental period and analyzed for zinc content, 67Zn isotope ratio, and dysprosium content. The mean (± SD) zinc intake from the representative Chinese diet was 10.6 ± 1.5 mg/d. The phytic acid-to-zinc molar ratio in the diet was 6.4. The absorption rate of 67Zn was 27.9% ± 9.2%. The RNI of zinc, which were calculated by the absorption rate in elderly men and women, were 10.4 and 9.2 mg/d, respectively. This study got the dietary Zn absorption in a Chinese elderly population. We found that Zn absorption was higher in elderly men than in elderly women. The current RNI in elderly female is lower than our finding, which indicates that more attention is needed regarding elderly females' zinc status and health.


Role of noncoding RNA in vascular remodelling.
Noncoding RNAs (ncRNAs), such as microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) are becoming fundamentally important in the pathophysiology relating to injury-induced vascular remodelling. We highlight recent studies that demonstrate the involvement of ncRNAs in vein graft disease, in in-stent restenosis and in pulmonary arterial hypertension, with a particular focus on endothelial cell and vascular smooth muscle cell function. We also briefly discuss the emerging role of exosomal-derived ncRNAs and how this mechanism impacts on vascular function. ncRNAs have been described as novel regulators in the pathophysiology of vascular injury, inflammation, and vessel wall remodelling. In particular, several studies have demonstrated that manipulation of miRNAs can reduce the burden of pathological vascular remodelling. Such studies have also shown that exosomal miRNA-mediated, cell-to-cell communication between endothelial cells and vascular smooth muscle cells is critical in the disease process. In addition to miRNAs, lncRNAs are emerging as regulators of vascular function in health and disease. Although lncRNAs are complex in both their sheer numbers and mechanisms of action, identifying their contribution to vascular disease is essential. Given the important roles of ncRNAs in vascular injury and remodelling together will their capacity for cell-to-cell communication, manipulating ncRNA might provide novel therapeutic interventions.


Pharmacokinetics of ceftiofur in healthy and lipopolysaccharide-induced endotoxemic newborn calves treated with single and combined therapy.
The aim of this research was to compare plasma pharmacokinetics of ceftiofur sodium (CS) in healthy calves, and in calves with experimentally induced endotoxemia. Six calves received CS (2.2 mg/kg, IM) 2 hr after intravenous administration of 0.9% NaCl (Ceft group). After a washout period, the same 6 calves received CS 2 hr after intravenous injection of lipopolysaccharide (LPS+Ceft group). Another group of 6 calves received a combination of drug therapies that included CS 2 hr after administration of 0.9% NaCl (Comb group). A third group of 6 calves received the same combination therapy regimen 2 hr after intravenous injection of lipopolysaccharide (LPS+Comb group). Plasma concentrations of CS and all desfuroylceftiofur-related metabolites were determined using HPLC, and its pharmacokinetic properties were determined based on a two-compartment model. The peak concentration of CS in the LPS+Comb group occurred the earliest, and the clearance rate of CS was the highest in the Comb and LPS+Comb groups (P<0.05). The elimination half-life of CS in the LPS+Ceft group was longer than that in the Ceft and Comb groups (P<0.05). The results of this study indicate that combined therapies and endotoxemic status may alter the plasma pharmacokinetics of CS in calves.


TRIM28 and β-actin identified via nanobody-based reverse proteomics approach as possible human glioblastoma biomarkers.
Malignant gliomas are among the rarest brain tumours, and they have the worst prognosis. Grade IV astrocytoma, known as glioblastoma multiforme (GBM), is a highly lethal disease where the standard therapies of surgery, followed by radiation and chemotherapy, cannot significantly prolong the life expectancy of the patients. Tumour recurrence shows more aggressive form compared to the primary tumour, and results in patient survival from 12 to 15 months only. Although still controversial, the cancer stem cell hypothesis postulates that cancer stem cells are responsible for early relapse of the disease after surgical intervention due to their high resistance to therapy. Alternative strategies for GBM therapy are thus urgently needed. Nanobodies are single-domain antigen-binding fragments of heavy-chain antibodies, and together with classical antibodies, they are part of the camelid immune system. Nanobodies are small and stable, and they share a high degree of sequence identity to the human heavy chain variable domain, and these characteristics offer them advantages over classical antibodies or antibody fragments. We first immunised an alpaca with a human GBM stem-like cell line prepared from primary GBM cultures. Next, a nanobody library was constructed in a phage-display vector. Using nanobody phage-display technology, we selected specific GBM stem-like cell binders through a number of affinity selections, using whole cell protein extracts and membrane protein-enriched extracts from eight different GBM patients, and membrane protein-enriched extracts from two established GBM stem-like cell lines (NCH644 and NCH421K cells). After the enrichment, periplasmic extract ELISA was used to screen for specific clones. These nanobody clones were recloned into the pHEN6 vector, expressed in Escherichia coli WK6, and purified using immobilised metal affinity chromatography and size-exclusion chromatography. Specific nanobody:antigen pairs were obtained and mass spectrometry analysis revealed two proteins, TRIM28 and β-actin, that were up-regulated in the GBM stem-like cells compared to the controls.


Enterovirus 71 induces mitochondrial reactive oxygen species generation that is required for efficient replication.
Redox homeostasis is an important host factor determining the outcome of infectious disease. Enterovirus 71 (EV71) infection has become an important endemic disease in Southeast Asia and China. We have previously shown that oxidative stress promotes viral replication, and progeny virus induces oxidative stress in host cells. The detailed mechanism for reactive oxygen species (ROS) generation in infected cells remains elusive. In the current study, we demonstrate that mitochondria were a major ROS source in EV71-infected cells. Mitochondria in productively infected cells underwent morphologic changes and exhibited functional anomalies, such as a decrease in mitochondrial electrochemical potential ΔΨ(m) and an increase in oligomycin-insensitive oxygen consumption. Respiratory control ratio of mitochondria from infected cells was significantly lower than that of normal cells. The total adenine nucleotide pool and ATP content of EV71-infected cells significantly diminished. However, there appeared to be a compensatory increase in mitochondrial mass. Treatment with mito-TEMPO reduced eIF2α phosphorylation and viral replication, suggesting that mitochondrial ROS act to promote viral replication. It is plausible that EV71 infection induces mitochondrial ROS generation, which is essential to viral replication, at the sacrifice of efficient energy production, and that infected cells up-regulate biogenesis of mitochondria to compensate for their functional defect.


Phase separation of lipid membranes analyzed with high-resolution secondary ion mass spectrometry.
Lateral variations in membrane composition are postulated to play a central role in many cellular events, but it has been difficult to probe membrane composition and organization on length scales of tens to hundreds of nanometers. We present a high-resolution imaging secondary ion mass spectrometry technique to reveal the lipid distribution within a phase-separated membrane with a lateral resolution of approximately 100 nanometers. Quantitative information about the chemical composition within small lipid domains was obtained with the use of isotopic labels to identify each molecular species. Composition variations were detected within some domains.


Simple and reliable CYP1A2 phenotyping by the paraxanthine/caffeine ratio in plasma and in saliva.
Several procedures to monitor CYP1A2 activity in vivo by the use of caffeine as a probe have been proposed. They comprise caffeine clearance, based on both plasma and saliva concentrations, urinary metabolite ratios, the 13C-caffeine breath test, and the paraxanthine/caffeine ratio in plasma. The latter method is fast, simple, economical and restricted to one sampling point. In this study, we retrospectively analysed four clinical trials comprising 78 subjects to validate the use of the paraxanthine/caffeine ratios in plasma and saliva for CYP1A2 activity. The validation was done by correlation of these ratios to the systemic caffeine clearance as a reference method. Additionally, urinary metabolite ratios and the caffeine breath test were included in the analysis. The paraxanthine/caffeine ratios in plasma and saliva preferably 5-7 h after administration of caffeine most closely resembled systemic caffeine clearance with correlation coefficients typically higher than r = 0.85. An equation to estimate systemic caffeine clearance from the paraxanthine/caffeine ratios taken at any time within 3-7 h postdose was developed. Correlations of systemic clearance with urinary metabolite ratios and the caffeine breath test were less reliable both in this investigation and in the literature. In conclusion, the paraxanthine/caffeine ratios in plasma and saliva appear a valid and inexpensive method of assessing CYP1A2 activity in vivo. Apparent distribution of CYP1A2 activity for all healthy subjects appeared bimodal in nonsmokers (n = 29) and smokers (n = 17).


In Vitro Evaluation of the Technological and Probiotic Potential of Pichia kudriavzevii Strains Isolated from Traditional Fermented Foods.
The isolation of endogenous yeast strains from traditionally fermented food products to use as functional starter cultures has become more popular for improved food safety, quality, and beneficial health effects. In this study, 107 Pichia kudriavzevii strains were isolated from sourdough, shalgam, tarhana, artisanal Tulum cheese, and yogurt. The strains were identified by DNA fingerprinting using iPBS-PCR method before technological and probiotic characterization. The multivariate statistical approach revealed that five strains were most promising in terms of technological characterization, including different harsh growth conditions. These strains were also examined in terms of probiotic properties with a commercial S. cerevisiae var boulardii MYA-796 strain. The multivariate statistical analyses indicated that P. kudriavzevii 5S5 were most promising in in vitro probiotic properties such as surviving in human GI conditions, adhering to intestinal cell lines, and exhibiting high hydrophobicity. Therefore, it seems to be a great starter candidate for the production of functional fermented food products.


Vitamin D deficiency and its relationship with cardiac iron and function in patients with transfusion-dependent thalassemia at Chiang Mai University Hospital.
Vitamin D deficiency is common in patients with thalassemia. Vitamin D deficiency could be related to cardiac dysfunction. Increased parathyroid hormone (PTH) is also known to be associated with heart failure. To determine the prevalence of Vitamin D deficiency and to explore the impact of Vitamin D deficiency on cardiac iron and function in patients with transfusion-dependent thalassemia. A cross-sectional study in patients with Transfusion-dependent thalassemia was conducted. Patients with liver disease, renal disease, type 1 diabetes, malabsorption, hypercortisolism, malignancy, and contraindication for MRI were excluded. Calcium, phosphate, PTH, vitamin D-25OH were measured. CardiacT2* and liver iron concentration (LIC) and left ventricular ejection fraction (LVEF) were determined. Results Sixty-one (33M/28F) patients with Transfusion-dependent thalassemia were enrolled. The prevalence of Vitamin D deficiency was 50.8%. Patients with cardiac siderosis had tendency for lower D-25OH than those without siderosis (15.9 (11.7-20.0) vs. 20.2 (15.85-22.3) ng/mL); p = 0.06). Serum calcium, phosphate, PTH, LIC, cardiac T2*, and LVEF were not different between the groups with or without Vitamin D deficiency. Patients with Vitamin D deficiency had significantly lower hemoglobin levels compared to those without Vitamin D deficiency (7.5 (6.93-8.33) vs. 8.1 (7.30-8.50) g/dL; p = 0.04). The median hemoglobin in the last 12 months was significantly correlated with D-25OH. Cardiac T2* had significant correlation with PTH. Vitamin D deficiency is prevalent in patients with Transfusion-dependent thalassemia. Vitamin D level is correlated with hemoglobin level. Vitamin D status should be routinely assessed in these patients. Low PTH is correlated with increased cardiac iron. This study did not demonstrate an association between Vitamin D deficiency and cardiac iron or function in patients with Transfusion-dependent thalassemia.


Theoretical Insights into the Selectivity of Hydrophilic Sulfonated and Phosphorylated Ligands to Am(III) and Eu(III) Ions.
The separation of lanthanides and actinides has attracted great attention in spent nuclear fuel reprocessing up to date. In addition, liquid-liquid extraction is a feasible and useful way to separate An(III) from Ln(III) based on their relative solubilities in two different immiscible liquids. The hydrophilic bipyridine- and phenanthroline-based nitrogen-chelating ligands show excellent performance in separation of Am(III) and Eu(III) as reported previously. To profoundly explore the separation mechanism, herein, we first of all designed four hydrophilic sulfonated and phosphorylated ligands L1, L2, L3, and L4 based on the bipyridine and phenanthroline backbones. In addition, we studied the structures of these ligands and their neutral complexes [ML(NO3)3] (M = Am, Eu) as well as the thermodynamic properties of complexing reactions through the scalar relativistic density functional theory. According to the changes of the Gibbs free energy for the back-extraction reactions, the phenanthroline-based ligands L2 and L4 have stronger complexing capacity for both Am(III) and Eu(III) ions while the phosphorylated ligand L3 with the bipyridine framework has the highest Am(III)/Eu(III) selectivity. In addition, the charge decomposition analysis revealed a higher degree of charge transfer from the ligand to Am(III), suggesting stronger donor-acceptor interactions in the Am(III) complexes. This study can provide theoretical insights into the separation of actinide(III)/lanthanide(III) using hydrophilic sulfonated and phosphorylated N-donor ligands.


Spatiotemporal cascade of transcription factor binding required for promoter activation.
Promoters often contain multiple binding sites for a single factor. The yeast HO gene contains nine highly conserved binding sites for the SCB (Swi4/6-dependent cell cycle box) binding factor (SBF) complex (composed of Swi4 and Swi6) in the 700-bp upstream regulatory sequence 2 (URS2) promoter region. Here, we show that the distal and proximal SBF sites in URS2 function differently. Chromatin immunoprecipitation (ChIP) experiments show that SBF binds preferentially to the left side of URS2 (URS2-L), despite equivalent binding to the left-half and right-half SBF sites in vitro. SBF binding at URS2-L sites depends on prior chromatin remodeling events at the upstream URS1 region. These signals from URS1 influence chromatin changes at URS2 but only at sites within a defined distance. SBF bound at URS2-L, however, is unable to activate transcription but instead facilitates SBF binding to sites in the right half (URS2-R), which are required for transcriptional activation. Factor binding at HO, therefore, follows a temporal cascade, with SBF bound at URS2-L serving to relay a signal from URS1 to the SBF sites in URS2-R that ultimately activate gene expression. Taken together, we describe a novel property of a transcription factor that can have two distinct roles in gene activation, depending on its location within a promoter.


Evaluation of Undescended Testes in Newborns: It Is Really Simple, Just Not Easy.
The evaluation of the testicular position in newborns is important to ensure timely initiation of therapy. The aim of our study was to assess the reliability of a routinely performed screening examination. Newborns were examined by a pediatrician between 48 and 72 h after birth. Boys with suspected cryptorchidism were double-checked by a pediatric urologist within 24 h. 1,181/2,353 children included in the study between June 2015 and December 2017 were male. Eight hundred sixty-one boys could be included in this analysis; 5.8% (n = 50) were diagnosed with undescended testis (UDT) by the pediatrician. 30/50 boys were double-checked at the Department of Pediatric Urology. Forty percent (20/50) were lost to follow-up. In 43% (13/30), the diagnosis could be confirmed. Three former studies had shown a relevant discrepancy in the results of the diagnosis of UDT made by health care providers and urologists/pediatric surgeons. To our knowledge, this is the first study evaluating the testicular position in male newborns in such a large prospective birth cohort study by physicians with ranging expertise within 1 day. Further treatment for UDT is based on clinical examination. Ours and previous studies can clearly show the various findings in boys suspected having UDT. Therefore, it is essential that the diagnosis is confirmed by a specialist before a therapy is initiated.


Antimicrobial properties of substituted salicylaldehydes and related compounds.
A systematic survey of the antimicrobial properties of substituted salicylaldehydes and some related aromatic aldehydes is reported. A total of 23 different compounds, each at four different concentrations, were studied using a panel of seven microbes (Aspergillus niger, Bacillus cereus, Candida albicans, Escherichia coli, Pseudomonas aeruginosa, Saccharomyces cerevisiae and Staphylococcus epidermidis) and employing the paper disc agar diffusion method. Several aldehydes, most notably halogenated, nitro-substituted and hydroxylated salicylaldehydes, displayed highly potent activity against the microbes studied, giving inhibitory zones up to 49 mm in diameter (paper disc diameter 6 mm), while unsubstituted benzaldehyde and salicylaldehyde had minimal activity. Further, 4,6-dimethoxysalicylaldehyde had considerable activity against C albicans and slight activity against S. cerevisiae, while displaying minimal activity against bacteria. Also two aromatic dialdehydes had interesting activity. In general, P. aeruginosa was the least sensitive microbe, a result that is in line with observations from a large screening project, in which this microbe was the one against which the least number of active substances was found. Interestingly, the structure-activity relationships of the aldehydes studied were clearly different for different microbes. Many of the aldehydes tested had such high antimicrobial activity that they are noteworthy candidates for practical applications as well as interesting lead compounds for the development of novel antimicrobial drugs and disinfectants. The structure-activity relationships are discussed in detail. For high activity, substituents are required in benzaldehyde as well as in its 2-hydroxy derivative salicylaldehyde. One hydroxy group alone (at the 2-position) is not enough, but further hydroxylation may produce high activity. The effects of substituents are in some cases dramatic. Halogenation, hydroxylation and nitro substitution may produce highly active compounds, but the effects are not easily predicted nor can they be extrapolated from one microbe to another.


Acute Diffusion-Weighted Imaging Signaling Severe Periventricular Leukomalacia in Preterm Infants: Case Report and Review of Literature.
Introduction: Periventricular leukomalacia occurs in up to 25% of very preterm infants resulting in adverse neurodevelopmental outcomes. In its acute phase, periventricular leukomalacia is clinically silent. Although ultrasonography is widely available, its sensitivity in the early detection of periventricular leukomalacia is low. Case Report and Published Literature: We identified a preterm infant with early diffusion-weighted imaging changes that later evolved to periventricular leukomalacia. Thirty-two cases of abnormal diffusion-weighted imaging reliably heralding severe periventricular leukomalacia in the preterm infant have been published in the literature. Notable features include the following: (1) infants were more mature preterm infants (29-36 weeks' gestation); (2) findings were often serendipitous with benign clinical courses; (3) diffusion-weighted imaging changes only were evident in the first weeks of life with later evolution to more classical abnormalities on conventional magnetic resonance imaging (MRI) or ultrasonography. Conclusion: Diffusion-weighted imaging in the first week of life may be a reliable early marker of severe periventricular leukomalacia injury in more mature preterm infants.


A general dimensionless equation of gas transport by high-frequency ventilation.
To identify a general relationship between eucapnic oscillatory flow (Vosc) and frequency (f) in high-frequency ventilation (HFV), we searched the literature for eucapnic HFV data in different mammalian species. We found suitable results for rat, rabbit, monkey, dog, human, and horse, which we expressed in terms of two dimensionless variables, Q = Vosc/Va and F = f/(VA/VD), with VA the alveolar ventilation and VD the volume of the conducting airways. The experimental HFV data define the linear regression equation in Q = 0.54 In F + 0.92 (R = 0.94). Krogh's equation for conventional ventilation (CV), Vosc = VA + fVD, in dimensionless terms becomes Q = 1 + F, which is valid for low F. The intersection of the CV and HFV equations at F = 5.0 defines a transition frequency, ft = 5.0 (VA/VD). At that point the alveolar ventilation per breath, VA/f, represents 20% of VD, and tidal volume (VT) equals 1.20 VD. For eucapnia ft ranges from 5.9 Hz in the rat to 0.9 Hz in the dog. The dimensional form of our HFV equation, VA = 0.13 (VT/VD)1.2 (VTf) is very similar to other empirical equations reported for dogs in noneucapnic settings. Therefore the dimensionless equation should also be valid within a species at noneucapnic settings.


Disease activity in rheumatoid arthritis is inversely related to cerebral TSPO binding assessed by [11C]PBR28 positron emission tomography.
Reumatoid Arthritis (RA) is an autoimmune disorder characterized by peripheral joint inflammation. Recently, an engagement of the brain immune system has been proposed. The aim with the current investigation was to study the glial cell activation marker translocator protein (TSPO) in a well characterized cohort of RA patients and to relate it to disease activity, peripheral markers of inflammation and autonomic activity. Fifteen RA patients and fifteen healthy controls matched for age, sex and TSPO genotype (rs6971) were included in the study. TSPO was measured using Positron emission tomography (PET) and the radioligand [11C]PBR28. The outcome measure was total distribution volume (VT) estimated using Logan graphical analysis, with grey matter (GM) as the primary region of interest. Additional regions of interest analyses as well as voxel-wise analyses were also performed. Clinical evaluation of disease activity, symptom assessments, serum analyses of cytokines and heart rate variability (HRV) analysis of 24 h ambulatory ECG were performed in all subjects. There were no statistically significant group differences in TSPO binding, either when using the primary outcome VT or when normalizing VT to the lateral occipital cortex (p > 0.05). RA patients had numerically lower VT values than healthy controls (Cohen's D for GM = -0.21). In the RA group, there was a strong negative correlation between [11C]PBR28 VT in GM and disease activity (DAS28)(r = -0.745, p = 0.002, corrected for rs6971 genotype). Higher serum levels of IFNγ and TNF-α were found in RA patients compared to controls (p < 0.05) and several measures of autonomic activity showed significant differences between RA and controls (p < 0.05). However, no associations between markers of systemic inflammation or autonomic activity and cerebral TSPO binding were found. In conclusion, no statistically significant group differences in TSPO binding as measured with [11C]PBR28 PET were detected. Within the RA group, lower cerebral TSPO binding was associated with higher disease activity, suggesting that cerebral TSPO expression may be related to disease modifying mechanisms in RA. In light of the earlier confirmed neuro-immune features of RA, these results warrant further investigations regarding neuro-immune joint-to-CNS signalling to open up for potentially new treatment strategies.


Subcutaneous versus intravenous formulation of trastuzumab for HER2-positive early breast cancer: updated results from the phase III HannaH study.
HannaH (NCT00950300) was a phase III, randomized, international, open-label study that compared pharmacokinetics (PK), efficacy, and safety of two different trastuzumab formulations [subcutaneous (s.c.) and intravenous (i.v.)] in HER2-positive, operable, locally advanced, or inflammatory breast cancer in the neoadjuvant/adjuvant setting. The co-primary end points, to show noninferiority of s.c. versus i.v. trastuzumab in terms of serum concentration (Ctrough) and pathologic complete response (pCR) were met; safety profiles were comparable at 12 months' median follow-up. Secondary end points included safety and tolerability, PK profile, immunogenicity, and event-free survival (EFS). We now report updated safety and efficacy data after a median follow-up of 20 months. Patients (N = 596) were treated with eight cycles of neoadjuvant chemotherapy, administered concurrently with 3-weekly s.c. trastuzumab (fixed dose of 600 mg) or the standard weight-based i.v. method. Following surgery, patients continued trastuzumab treatment to complete 1 year of therapy. Updated analyses of PK, efficacy, safety, and immunogenicity data were carried out. s.c. trastuzumab was generally well tolerated and the incidence of adverse events (AEs), including grade 3 or 4 AEs, between treatment groups was comparable. A slightly higher incidence of serious AEs (SAEs), mainly due to infections, was reported with s.c. treatment {64 [21.5%; 95% confidence interval (CI) 17.0%-26.7%] versus 42 (14.1%; 95% CI 10.4%-18.6%) in the i.v. group}; however, the differences were small and often based on rare events, with no observable pattern across reported events. An early analysis of EFS showed rates of 95% in both groups 1 year postrandomization. Exploratory analyses did not reveal an association between toxicity and body weight or exposure. Overall, the safety profile of s.c. trastuzumab was consistent with the previously published data from HannaH and the known safety profile of i.v. trastuzumab. EFS rates were comparable between the i.v. and s.c. groups. NCT00950300.


Metformin Treatment Leads to Increased HIV Transcription and Gene Expression through Increased CREB Phosphorylation and Recruitment to the HIV LTR Promoter.
Antiretroviral therapy has effectively suppressed HIV infection and replication and prolonged the lifespan of HIV-infected individuals. In the meantime, various complications including type 2 diabetes associated with the long-term antiviral therapy have shown steady increases. Metformin has been the front-line anti-hyperglycemic drug of choice and the most widely prescribed medication for the treatment of type 2 diabetes. However, little is known about the effects of Metformin on HIV infection and replication. In this study, we showed that Metformin treatment enhanced HIV gene expression and transcription in HIV-transfected 293T and HIV-infected Jurkat and human PBMC. Moreover, we demonstrated that Metformin treatment resulted in increased CREB expression and phosphorylation, and TBP expression. Furthermore, we showed that Metformin treatment increased the recruitment of phosphorylated CREB and TBP to the HIV LTR promoter. Lastly, we showed that inhibition of CREB phosphorylation/activation significantly abrogated Metformin-enhanced HIV gene expression. Taken together, these results demonstrated that Metformin treatment increased HIV transcription, gene expression, and production through increased CREB phosphorylation and recruitment to the HIV LTR promoter. These findings may help design the clinical management plan and HIV cure strategy of using Metformin to treat type 2 diabetes, a comorbidity with an increasing prevalence, in people living with HIV.


Study of the Relationship between Polymorphisms in the IL-8 Gene Promoter Region and Coccidiosis Resistance Index in Jinghai Yellow Chickens.
Interleukin 8 (IL-8) participates in the immune response and has the function of inducing neutrophils to release lysosomal enzymes and eliminate pathogens. This study was to investigate the effect of single nucleotide mutations in the IL-8 gene promoter region on the coccidiosis resistance index. In this study, 180 infected Eimeria tenella (E. tenella) Jinghai yellow chickens were used as experimental samples. DNA sequencing technology was used to detect single nucleotide polymorphisms (SNPs) in the IL-8 gene promoter region. The association between these SNPs and coccidiosis resistance indexes (including superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-PX), catalase (CAT), nitric oxide (NO), interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-6 (IL-6), IL-8, and interferon-γ (IFN-γ)) were analyzed. Three SNPs (T-550C, G-398T, and T-360C) were detected. Significant associations were found between each genotype at the T-550C site with NO (p-value = 0.006) and IL-8 (p-value = 0.034) indexes. Significant associations were found between each genotype at the G-398T site with SOD (p-value = 0.042), CAT (p-value = 0.049), NO (p-value = 0.008), and IL-2 (p-value = 0.044) indexes. Significant associations were found between each genotype at the T-360C site with SOD (p-value = 0.007), NO (p-value = 0.046), IL-2 (p-value = 0.041), IL-8 (p-value = 0.039), and IFN-γ (p-value = 0.042) indexes. Haplotype analysis showed that multiple indexes of the H1H3 haplotype combination were significantly higher than other haplotype combinations. Therefore, mutation of the IL-8 gene promoter region has a significant regulatory effect on the coccidiosis resistance index, with a change in transcription factor binding potentially altering IL-8 gene expression, thereby further affecting the IL-8 level in plasma. However, the specific mechanism needs further study.


Idiopathic peripheral gangrene in Nigeria.
A severe, acutely evolving peripheral gangrene of unknown etiology has been reported sporadically from different parts of Africa. This case report describes such a case of fulminant gangrene of the extremities following a febrile diarrheal illness associated with abdominal pain, polyarthropathy, and altered hemostatic function in a black adolescent male. A model of autoimmune causation for this uncommon clinical syndrome is proposed with guidelines for therapy.


[Chronic graft-versus-host disease with multiple serositis after bone marrow transplantation from non-inherited maternal antigen-complementary sibling donor].
We report a patient who developed multiple serositis during chronic graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation from a non-inherited maternal antigen (NIMA) -complementary sibling donor. The patient was a 9-year-old boy with myelodysplastic syndrome, who urgently underwent bone marrow transplantation from his NIMA-complementary HLA two-locus-mismatched sister following graft failure of cord blood transplantation. Engraftment was successfully confirmed and no acute GVHD developed. After withdrawal of tacrolimus to prevent recurrent viral infection, he developed pleural effusion, ascites and edema approximately 6 months after transplantation. His clinical symptoms were resolved by methylprednisolone pulse therapy, but he subsequently progressed to develop pericardial effusion, pneumothorax and truncal panniculitis. Pleural and pericardial effusion contained numerous lymphocytes, which gradually subsided with continuous drainage. His symptoms were thereafter controlled by the addition of mycophenolate mofetil (MMF) administration, and his current performance status is almost perfect by the administration of prednisolone (5 mg/day) and MMF at 6 years after transplantation. Although multiple serositis associated with GVHD is known to have a poor prognosis, the multiple symptoms of this patient improved gradually, probably owing to a lack of acute GVHD and the effect of MMF.


CD40 and IL-4 regulate murine CD27L expression.
It is well known that interactions between accessory molecules on T cells and their ligands on APC play a key role in regulating T cell effector activity. The factors controlling the expression of these molecules are thus important determinants in the outcome of T cell activation. We have examined the expression of the murine ligand for CD27, a costimulatory molecule on T cells. Evidence is shown that CD27L is expressed at a low level on resting B cells but not on T cells, and that activation of B cells by culture with LPS or anti-IgM Ab increases the expression of CD27L. Interestingly, coligation of CD40 down-regulates CD27L on LPS-activated B cells but not on anti-Ig-activated cells. These findings suggest that costimulation via the CD27-CD27L pathway may be limited to interactions involving Ag-specific B cells, i.e., B cells specifically activated via their Ig receptors. In addition, testing a spectrum of different cytokines indicated that IL-4 and TGF, but not IL-2, IL-10, or IFN-gamma, prevented up-regulation of CD27L expression on activated B cells even when activation was induced by Ig signaling. The capacity of IL-4 to prevent CD27L expression could thus serve to limit CD27-CD27L interactions to Th1-type T cell responses.


Use of a newly established human cell line (SU-CCS-1) to demonstrate the relationship of clear cell sarcoma to malignant melanoma.
A new tumor cell line, designated SU-CCS-1, was established from the malignant pleural effusion of a 16-year-old Caucasian girl with clear cell sarcoma. Morphological studies at the light- and electron-microscopic levels revealed similar features between the SU-CCS-1 cells and the primary tumor. Ultrastructural and cytochemical techniques showed that both the SU-CCS-1 cell line and the original tumor were amelanotic in nature. The malignant derivation of the SU-CCS-1 cell line was demonstrated by intracranial and s.c. heterotransplantation in the nude, athymic mouse and by cytogenetic analysis which showed that the cell line had a hypodiploid chromosome number and several karyotypic abnormalities. Live-cell radioimmunoassay procedures using a large panel of monoclonal antibodies directed against tumor-associated antigens revealed that, phenotypically, SU-CCS-1 closely resembled melanoma tumor cell lines. Immunological assays for the detection of neuroendocrine-associated peptides, hormones, and enzymes revealed that, like melanoma, the SU-CCS-1 cell line was actively producing alpha-melanotropin, S-100 antigen, and nerve growth factor. A notable difference between these tumor types was the capacity of SU-CCS-1 to produce bombesin, an active neuropeptide whose synthesis has been found in cell lines from patients with small cell carcinoma of the lung. From these studies, we concluded that the SU-CCS-1 cell line is phenotypically similar to melanoma, yet displays unique characteristics which distinguishes it from other sarcomas. The availability of an established clear cell sarcoma cell line will greatly facilitate further studies aimed at uncovering the histogenesis of this rare cancer.


Aluminium allergy in patients hyposensitized with aluminium-precipitated antigen extracts.
During hyposensitization therapy with aluminium-precipitated antigen solutions, a small % of patients develop persistent subcutaneous nodules at the injection site; the existence of delayed sensitivity to aluminium has been implicated in the pathogenesis of these nodules. We studied the prevalence of aluminium sensitivity (using patch, prick and intradermal tests) and common contact allergens (TRUE Test) in 20 healthy subjects, and in 40 patients treated with aluminium-containing extracts, 20 of whom had persistent subcutaneous nodules that remained for more than 2 months, the other half having no nodular reactions or nodules that remained for less than 2 months. Aluminium sensitivity was found only in those patients of the treated group who had persistent nodular reactions, 4 cases of positivity to an aluminium chloride patch test being found. All 4 cases were women, nodules remained for more than 6 months, and intracutaneous tests were negative. 3 of them also had contact sensitivity to nickel. In 2 cases, nodules were removed for histological and histochemical examination, showing non-specific inflammatory granulomas, and aluminium crystals being found in only 1 case. It is concluded that delayed sensitivity to aluminium appears to be implicated in the pathogenesis of persistent nodular reactions, but sensitivity to aluminium was not found in patients treated with aluminium-precipitated extracts without persistent nodular reactions.


Diabetic encephalopathy-related depression: experimental evidence that insulin and clonazepam restore antioxidant status in rat brain.
There is increasing evidence suggesting that oxidative stress plays an important role in the development of many chronic and degenerative conditions such as diabetic encephalopathy and depression. Considering that diabetic rats and mice present higher depressive-like behaviour when submitted to the forced swimming test and that treatment with insulin and/or clonazepam is able to reverse the behavioural changes of the diabetic rats, the present work investigated the antioxidant status, specifically total antioxidant reactivity and antioxidant potential of insulin and clonazepam, as well as the effect of this drugs upon protein oxidative damage and reactive species formation in cortex, hippocampus and striatum from diabetic rats submitted to forced swimming test. It was verified that longer immobility time in diabetic rats and insulin plus clonazepam treatment reversed this depressive-like behaviour. Moreover, data obtained in this study allowed to demonstrate through different parameters such as protein carbonyl content, 2'7'-dichlorofluorescein oxidation, catalase, superoxide dismutase, glutathione peroxidase assay, total radical-trapping antioxidant potential and total antioxidant reactivity that there is oxidative stress in cortex, hippocampus and striatum from diabetic rats under depressive-like behaviour and highlight the insulin and/or clonazepam effect in these different brain areas, restoring antioxidant status and protein damage.


Postabsorptive retinyl palmitate removal is retarded in lecithin-cholesterol acyltransferase deficiency.
Postabsorption fat clearance of intestinal lipoproteins indicated by retinyl palmitate was studied in two siblings with the classical lecithin-cholesterol acyltransferase (LCAT) deficiency and in a control group of 21 healthy subjects with similar apoprotein E 3/3 phenotype. Relatively high pre- and postabsorptive cholesterol esterification percentage of chylomicrons suggested that acyl coenzyme A:cholesterol acyltransferase activity was not inhibited. Postabsorptive levels of plasma lipids increased and decreased roughly similarly in the cases and controls with the postabsorptive peak values at about 4 h. Plasma total and chylomicron levels of retinyl palmitate were not affected by LCAT deficiency, while the removal of the vitamin from very low and intermediate density lipoproteins was clearly reduced so that the peak concentrations, values under the response curves and the time of the peak concentrations were markedly higher than in the controls. The long-lasting circulation of chylomicron remnants of density < 1.006-1.019 g ml-1 may have some clinical significance because postabsorptive lipoproteins are suspected to have atherogenic potentiality.


Whistles of the pantropical spotted dolphin (Stenella attenuata) in Santos Basin, western South Atlantic Ocean.
The vocal repertoire of the pantropical spotted dolphin (Stenella attenuata) is poorly documented, with no published information about acoustic signals from South Atlantic Ocean populations. We conducted passive acoustic monitoring and recording of S. attenuata population in the Santos Basin, Brazil, using a towed hydrophone array during line-transects surveys. Our monitoring yielded whistle samples derived from eight groups of S. attenuata, from which we selected 155 whistles for further analysis. Approximately 48% of the whistles presented ultrasonic frequency values, with maximum frequencies up to 31.1 kHz. Across the sample, the number of steps ranged from 0 to 20 and inflection points ranged from 0 to 8. On average, end frequencies were higher than start frequencies, and whistles generally presented wide frequency ranges, with an average of 11.3 kHz. The most predominant whistle contour category was "ascending-descending." Our study provides new information regarding the acoustic repertoire of this poorly documented species and will aid efforts for using acoustics to identify and monitor cetaceans in this region.


Acute intravenous and inhalation pharmacokinetics of 2,4-pentanedione in the Fischer 344 rat.
2,4-Pentanedione (2,4-PD; CAS No. 123-54-6), an industrial chemical, was investigated for its comparative pharmacokinetics in male Fischer 344 rats by a single intravenous (i.v.) injection of (4.3, 43, 148.5, and 430 mg/kg), or a 6-hr nose-only inhalation exposure (400 ppm) to 14C-2,4-PD. For the i.v. route, the plasma concentration of 14C-2,4-PD-derived radioactivity declined in a biexponential fashion. The overall form of the 14C plasma concentration-time curves and derived pharmacokinetic parameters indicated that dose-linear kinetics occurred in the i.v. dose range 4.3-148.5 mg/kg, but not with 430 mg/kg. Metabolism of 2,4-PD was quite rapid as the concentration of unmetabolized 2,4-PD declined steadily to undetectable after 8 hr. 14C-2,4-PD derived radioactivity was eliminated mainly as 14CO2 and in urine. For the 4.3, 43 and 148.5 mg/kg doses 14CO2 elimination was relatively constant (36.8, 38.8 and 42.3% in 48 hr samples respectively) and greater than urinary excretion (17.9, 14.3 and 29.6%; 48 hr specimens). At 430 mg/kg i.v. there was a reversal of the excretion pattern, with urine 14C excretion (54.7%) becoming greater than that for 14CO2 (27.3%). Excretion in expired volatiles and feces was small. Radiochromatograms of urine showed free 2,4-PD in the 12 hr sample, together with 7 other metabolites. Free 2,4-PD and 6 of the metabolites decreased or were not detectable in a 24 or 48 hr urine sample, but one peak (retention 7.9 min) increased progressively to become the major fraction (97%). Nose-only exposure to 400 ppm 14C-2, 4-PD produced a mean decrease in breathing rate of 20.1%, which was constant and sustained throughout exposure, due to a lengthening of the expiratory phase of the respiratory cycle. 14C-2,4-PD was rapidly absorbed during the first 3 hr of exposure, then began to plateau, but did not reach a steady state. Postexposure elimination of 14C from plasma followed a biexponential form with a t1/2 for the terminal disposition phase of 30.72 hr. Plasma unmetabolized 2,4-PD was present throughout the whole of the exposure phase, but was significantly less than total 14C. Postexposure, plasma unmetabolized 2,4-PD declined rapidly to undetectable concentrations by 12 hr. Radiolabel excretion was approximately equivalent in urine (37.6%) and expired 14CO2 (36.3%). Urine radiochromatograms showed a minor 2,4-PD contaminant (0.6-5.9% over 48 hr), along with 7 other peaks probably representing metabolites. As with the 148.5 mg/kg i.v. dose, the major metabolite peak was at 7.8 min retention, increasing from 41.1% (12 hr) to 62.8% (48 hr). Immediately postexposure, radioactivity was present in all tissues examined, but on a concentration basis (microgram equiv/g) there was no preferential accumulation of 14C in any tissue or organ. On a total organ basis, highest contents were in liver and kidney, presumably related to the metabolism and excretion of 2,4-PD. By 48 hr postexposure, concentrations had decreased in all tissues except fat, presumably due to the lipophilicity of 14C residues. The profile of the plasma-time radioactivity curves, and the presence of residual radioactivity in tissues at 48 hr postexposure, suggests that a cumulative process could occur with frequent repeated exposures.


Translation and Validation of Modified Dental Anxiety Scale: The Nepali Version.
Introduction. For proper management of anxious dental patients it is imperative to assess their levels of dental anxiety before treatment. Modified Dental Anxiety Scale (MDAS) is the most commonly used questionnaire to assess dental anxiety. But a Nepali version of MDAS is still lacking. Hence, the objective of this study was to develop a reliable and valid Nepali version of MDAS. Materials and Methods. The English version of the MDAS was translated into Nepali following a forward and backward translation process. Following pretesting and cognitive interviewing a final version of Nepali questionnaire was obtained. One hundred and fifty patients attending Department of Orthodontics completed the Nepali version of MDAS questionnaire at their convenience. Also, patients were asked to rate their overall anxiety on a 100 mm visual analog scale (VAS). A test-retest of the questionnaire was performed with 30 patients after 2 weeks. Results. Cronbach's alpha value of the Nepali version of MDAS was 0.775. The Intraclass Correlation Coefficient between test and retest was 0.872. Spearman's correlation coefficient between the total MDAS score and VAS score was 0.838. Conclusion. The translated Nepali version of MDAS is a reliable and valid instrument to measure the dental anxiety of Nepali patients.


Truncated recombinant human SP-D attenuates emphysema and type II cell changes in SP-D deficient mice.
Surfactant protein D (SP-D) deficient mice develop emphysema-like pathology associated with focal accumulations of foamy alveolar macrophages, an excess of surfactant phospholipids in the alveolar space and both hypertrophy and hyperplasia of alveolar type II cells. These findings are associated with a chronic inflammatory state. Treatment of SP-D deficient mice with a truncated recombinant fragment of human SP-D (rfhSP-D) has been shown to decrease the lipidosis and alveolar macrophage accumulation as well as production of proinflammatory chemokines. The aim of this study was to investigate if rfhSP-D treatment reduces the structural abnormalities in parenchymal architecture and type II cells characteristic of SP-D deficiency. SP-D knock-out mice, aged 3 weeks, 6 weeks and 9 weeks were treated with rfhSP-D for 9, 6 and 3 weeks, respectively. All mice were sacrificed at age 12 weeks and compared to both PBS treated SP-D deficient and wild-type groups. Lung structure was quantified by design-based stereology at the light and electron microscopic level. Emphasis was put on quantification of emphysema, type II cell changes and intracellular surfactant. Data were analysed with two sided non-parametric Mann-Whitney U-test. After 3 weeks of treatment, alveolar number was higher and mean alveolar size was smaller compared to saline-treated SP-D knock-out controls. There was no significant difference concerning these indices of pulmonary emphysema within rfhSP-D treated groups. Type II cell number and size were smaller as a consequence of treatment. The total volume of lamellar bodies per type II cell and per lung was smaller after 6 weeks of treatment. Treatment of SP-D deficient mice with rfhSP-D leads to a reduction in the degree of emphysema and a correction of type II cell hyperplasia and hypertrophy. This supports the concept that rfhSP-D might become a therapeutic option in diseases that are characterized by decreased SP-D levels in the lung.


Experimental determining the coherent-mode structure of vector electromagnetic field through its decomposition in reference basis.
A technique for experimental determining the coherent-mode structure of electromagnetic field is proposed. This technique is based on the coherence measurements of the field in some reference basis and represents a nontrivial vector generalization of the dual-mode field correlation method recently reported by F. Ferreira and M. Belsley [Opt. Lett.38(21), 4350 (2013)]. The justifiability and efficiency of the proposed technique is illustrated by an example of determining the coherent-mode structure of some specially generated and experimentally characterized secondary electromagnetic source.


Pharmacokinetic aspects of habituation to benzodiazepines.
In state of tolerance to the sedative effect of nitrazepam (NTZ) its pharmacokinetic properties are changed: the absorption is slowed down, the elimination, in contrast, is accelerated due to the rapid biotransformation. The NTZ content in brain tissue is increased significantly with respect to the brain levels of animals treated with a single dose of NTZ. In animals of only moderate intensity of tolerance produced by oxazepam (OX) the brain concentrations of OX are correlated with the developed tolerance.


How do we choose the appropriate animal model for antiseizure therapy development?
Introduction: Epilepsy affects over 65 million people worldwide, and despite the numerous therapies that are currently available for the symptomatic management of chronic seizures, a substantial proportion of the population has not achieved adequate seizure control. Developing more effective and better-tolerated therapies will benefit patients worldwide. Areas covered: This article will discuss the relevant preclinical models that have been instrumental to the development of over 20 antiseizure drugs (ASDs) currently on the market today. While there have been meaningful therapies already developed over the last several decades, this article will highlight remaining areas of unmet medical need. Innovative models of pharmacoresistant epilepsy may advance therapies for patients who currently do not attain sufficient seizure control in the absence of adverse effects. There also remains a need to identify improved therapies for special patient populations, including the very young and old. Expert opinion: ASD development will still find utility in the established models that have been instrumental to the identification of impactful therapies. However, there should now be greater emphasis to implement those models in young and aged rodents to advance novel therapies for patients who are still in need of better tolerated or more effective therapies, such as pediatric and elderly patients.


Distracted by pleasure: Effects of positive versus negative valence on emotional capture under load.
We report 3 experiments examining the effects of positive versus negative valence and perceptual load in determining attention capture by irrelevant emotional distractors. Participants performed a letter search task searching for 1 of 2 target letters (X or N) in conditions of either low perceptual load (circular nontarget letters) or high perceptual load (angular nontarget letters that are similar to the target letters). On 25% of the trials an irrelevant emotional distractor was presented at the display center and participants were instructed to ignore it. The distractor stimulus was either positive or negative and was selected from 3 different classes: IAPS pictures of erotica or mutilated bodies (Experiment 1), happy or angry faces (Experiment 2), and faces associated with gain or loss in a prior value-learning phase involving a betting game (Experiment 3). The results showed a consistent pattern of interaction of load and valence across the 3 experiments. Irrelevant emotional distractors produced interference effects on search reaction time (RT) in conditions of low load, with no difference between negative and positive valence. High perceptual load, however, consistently reduced interference from the negative-valence distractors, but had no effect on the positive-valence distractors. As these results were consistently found across 3 different categories of emotional distractors, they suggest the general conclusion that attentional capture by irrelevant emotional distractors depends on both their valence and the level of perceptual load in the task and highlight the special status of distractors associated with pleasure.


Reactivity in the MEM-test of cancer patients' lymphocytes incubated with brain-derived preparations and extracts from malignant teratomas.
A batch of human encephalitogenic protein (HEP) was compared to preparations of microtubulin and associated proteins (MST) from guinea pig brain by serological techniques and the macrophage electrophoretic mobility (MEM) method. HEP consisted mainly of A1 protein and MST showed the characteristic double band in the 55,000 MW region and some additional weak protein bands. No cross-reactivity could be detected between HEP and MST by rabbit hyperimmune sera to these substances. The MEM test, however, revealed human lymphocyte reactivity to HEP and MST in both cancer and multiple sclerosis patients, but no remarkable responses in other patients. A synthetic nonapeptide (114-122 region of A1 protein) led to lymphokine release in 2 of 5 multiple sclerosis patients, in 1 of 13 cancer patients, and in none out of 13 control individuals. From these findings it is concluded that a minor component (contaminant?) present in both HEP and MST might induce lymphocyte responses in cancer patients. The A1 protein of HEP preparations is, if at all, not the only protein responsible for the so-called HEP-response of cancer patients. Continuing the MEM test experiments with extracts from single organ cancers, it could be shown that patients with malignant teratomas respond to a more or less broad spectrum of extracts bearing organ cancer specificity. On the other side, extracts made from 2 malignant teratoma tissues led to MEM responses in 8 of 13 cancers, in 2 of 11 control persons and in none of 5 multiple sclerosis patients. These findings underline the concept that tumour-associated antigens (possibly oncofetal antigens) do exist, which bear organ cancer specificity and will lead to a specific sensitization of the host. Such antigens may be expressed in teratomas in different qualities and amounts depending on the tissue differentiation capacity of a given teratoma.


Aeromonas septicemia in infants and children.
Our experience at the Ramathibodi Hospital with 20 infants and children who had Aeromonas septicemia is reviewed. Their ages were from 1 day to 14 years. Eighteen patients had underlying diseases: leukemia, 5; aplastic anemia, 4; cirrhosis, 2; thalassemia/hemoglobinopathy, 3; renal failure, 1; ileal perforation, 1; marasmus, 1; and cavernous hemangioma with thrombocytopenia, 1. Blood cultures yielded Aeromonas hydrophila in all patients, and four patients had polymicrobial bacteremia. Fifteen episodes of septicemia were community-acquired and five were hospital-acquired. The clinical manifestations of these patients were similar to septicemia due to other Gram-negative enteric bacilli. Two patients each had ecthyma gangrenosum, necrotizing fasciitis and meningitis. Antibiotic treatment included penicillins, cephalosporins, aminoglycosides and sulfamethoxazole-trimethoprim. The overall case fatality rate was 50%; eight of the nine patients with acute leukemia or aplastic anemia died. With the exception of one child the blood cultures were sterile in all patients before death. Aeromonas septicemia is an uncommon but severe infection which occurs predominantly in compromised hosts.


Bone Density, Osteocalcin and Deoxypyridinoline for Early Detection of Osteoporosis in Obese Children.
This study aimed at comparing between bone density using DEXA, serum osteocalcin and urinary DPD in obese and non obese prepubertal children. After taking the consent of eighty children they were subjected to: full examination, anthropometric measurements, blood samples were withdrawn to determine serum osteocalcin, Ca, Ph, alkaline phosphatase, and urinary DPD. Bone densities, body composition of the whole body were examined using DEXA. Data were analyzed using SPSS. All anthropometric variables showed significant increase in obese children except for height in comparison to control group. Total mass, lean + BMC, lean, fat, area, BMC, BMD and Z score of the whole body were significantly increased in obese children. Serum calcium showed significant increase while alkaline phosphatase was significantly decreased in obese children. DPD showed no significant difference between obese and non obese children. Significant negative correlation was found between ca, lean, lean + BMC and total mass. Serum alkaline phosphatase showed also a significant negative correlation with (lean + BMC and total mass). Serum osteocalcin showed negative significant correlation with area, BMC, BMD, lean and Z score. Obese children showed significant increase in anthropometric and DEXA parameters, increase in serum calcium and significant decrease in serum alkaline phosphatase. Osteocalcin was negatively correlated with most of DEXA results.


Leaf thickness of barley: genetic dissection, candidate genes prediction and its relationship with yield-related traits.
In this first genetic study on assessing leaf thickness directly in cereals, major and environmentally stable QTL were detected in barley and candidate genes underlying a major locus were identified. Leaf thickness (LT) is an important characteristic affecting leaf functions which have been intensively studied. However, as LT has a small dimension in many plant species and technically difficult to measure, previous studies on this characteristic are often based on indirect estimations. In the first study of detecting QTL controlling LT by directly measuring the characteristic in barley, large and stable loci were detected from both field and glasshouse trials conducted in different cropping seasons by assessing a population of 201 recombinant inbred lines. Four loci (locating on chromosome arms 2H, 3H, 5H and 6H, respectively) were consistently detected for flag leaf thickness (FLT) in each of these trials. The one on 6H had the largest effect, with a maximum LOD 9.8 explaining up to 20.9% of phenotypic variance. FLT does not only show strong interactions with flag leaf width and flag leaf area but has also strong correlations with fertile tiller number, spike row types, kernel number per spike and heading date. Though with reduced efficiency, these loci were also detectable from assessing second last leaf of fully grown plants or even from assessing the third leaves of seedlings. Taking advantage of the high-quality genome assemblies for both parents of the mapping population used in this study, three candidate genes underlying the 6H QTL were predicted based on orthologous analysis. These results do not only broaden our understanding on genetic basis of LT and its relationship with other traits in cereal crops but also form the bases for cloning and functional analysis of genes regulating LT in barley.


The clinical consequences of variable selection in multiple regression models: a case study of the Norwegian Opioid Maintenance Treatment program.
Background: Selecting which variables to include in multiple regression models is a pervasive problem in medical research.Objectives: Based on questionnaire data (n = 18538, 69.9% men) from the Norwegian Opioid Maintenance Treatment Program, this study aims to compare the performance of different variable selection methods and the potential clinical consequences of choice of method. The effect of missing data is also explored.Methods: The dependent variable was engagement in criminal behavior while in treatment. Twenty-nine potential covariates on demographics, psychosocial factors and drug use were tested for inclusion in a multiple logistic regression model. Both complete case and multiply imputed data were considered. We compared the results from variable selection methods ranging from expert-based and purposeful variable selection, through stepwise methods, to more recently developed penalized regression using the Least Absolute Shrinkage and Selection Operator (LASSO).Results: The various variable selection methods resulted in regression models including from 9 to 22 covariates. The stepwise selection procedures generated the models with the most covariates included. The choice of variable selection method directly affected the estimated regression coefficients, both in effect size and statistical significance. For several variables the expert-based approach disagreed with all data-driven methods.Conclusions: The choice of variable selection method may strongly affect the resulting regression model, along with accompanying effect sizes and confidence intervals. This may affect clinical conclusions. The process should consequently be given sufficient consideration in model building. We recommend combining expert knowledge with a data-driven variable selection method to explore the models' robustness.


Relationships between adiponectin, sex hormone binding globulin and insulin resistance in hyperthyroid Graves' disease women.
Adiponectin and sex hormone binding globulin (SHBG) play a role in glucose metabolism. Hyperthyroidism has an impact on carbohydrate metabolism and could affect insulin resistance. The aim of this study was to assess the associations between insulin resistance, adiponectin and SHBG among hyperthyroid Graves` disease (GD) women. The study was undertaken in 60 women with hyperthyroidism in the course of GD; 32 healthy women matched by BMI and age formed the control group. The concentrations of: free thyroxine (fT4), free triiodothyronine (fT3), thyroid-stimulating hormone (TSH), SHBG, insulin, adiponectin and glucose were measured, and the homeostasis model assessment (HOMA-I) was calculated. Compared to euthyroid subjects, hyperthyroid GD women had elevated glucose, fT4, fT3, adiponectin and SHBG concentrations, but there were no differences in HOMA-I. When we explored the relations between adiponectin as well as SHBG with glucose and HOMA-I, we observed that HOMA-I was associated with adiponectin and SHBG only in the control group, and in hyperthyroidism there were no such connections. We found positive associations between adiponectin, SHBG, fT4 and fT3 in the GD group. Elevated concentrations of adiponectin and SHBG were observed in hyperthyroidism but they were not related to insulin resistance.


Control group studies of suicide survivors: a review and critique.
Empirical studies of suicide survivors with designs that include control groups are reviewed. Investigations are discussed individually with respect to those in which participating survivors are friends or a combination of many different relationships to the deceased, parents of the deceased child, and spouses. Following the review is a brief discussion of the commonalities of the findings and the most frequent methodological difficulties. Concluding comments suggest methodological improvements as well as issues and groups for which additional investigations are needed.


Zinc deficiency in rats is associated with up-regulation of hippocampal NMDA receptor.
Data indicated that zinc deficiency may contribute to the development of depression; however changes induced by zinc deficiency are not fully described. In the present paper we tested whether the dietary zinc restriction in rats causes alterations in N-methyl-D-aspartate receptor (NMDAR) subunits in brain regions that are relevant to depression. Male Sprague Dawley rats were fed a zinc adequate diet (ZnA, 50 mg Zn/kg) or a zinc deficient diet (ZnD, 3 mg Zn/kg) for 4 or 6weeks. Then, the behavior of the rats was examined in the forced swim test, sucrose intake test and social interaction test. Western blot assays were used to study the alterations in NMDAR subunits GluN2A and GluN2B and proteins associated with NMDAR signaling in the hippocampus (Hp) and prefrontal cortex (PFC). Following 4 or 6 weeks of zinc restriction, behavioral despair, anhedonia and a reduction of social behavior occurred in rats with concomitant increased expression of GluN2A and GluN2B and decreased expression of the PSD-95, p-CREB and BDNF protein levels in the Hp. The up-regulation of GluN2A protein was also found in the PFC, but only after prolonged (6 weeks) zinc deprivation. The procedure of zinc restriction in rats causes behavioral changes that share some similarities to the pathophysiology of depression. Obtained data indicated that depressive-like behavior induced by zinc deficiency is associated with the changes in NMDAR signaling pathway.


Phase-locked coordination between two rhythmically active feeding structures in the mollusk Clione limacina. I. Motor neurons.
Coordination between different motor centers is essential for the orderly production of all complex behaviors, in both vertebrates and invertebrates. The current study revealed that rhythmic activities of two feeding structures of the pteropod mollusk Clione limacina, radula and hooks, which are used to extract the prey from its shell, are highly coordinated in a phase-dependent manner. Hook protraction always coincided with radula retraction, while hook retraction coincided with radula protraction. Thus hooks and radula were always moving in the opposite phases, taking turns grabbing and pulling the prey tissue out of the shell. Identified buccal ganglia motor neurons controlling radula and hooks protraction and retraction were rhythmically active in the same phase-dependent manner. Hook protractor motor neurons were active in the same phase with radula retractor motor neurons, while hook retractor motor neurons burst in phase with radula protractor motor neurons. One of the main mechanisms underlying the phase-locked coordination was electrical coupling between hook protractor and radula retractor motor neurons. In addition, reciprocal inhibitory synaptic connections were found between hook protractor and radula protractor motor neurons. These electrical and inhibitory synaptic connections ensure that rhythmically active hooks and radula controlling motor neurons are coordinated in the specific phase-dependent manner described above. The possible existence of a single multifunctional central pattern generator for both radula and hook motor centers is discussed.


In vitro maturation is slowed in prepubertal lamb oocytes: ultrastructural evidences.
In vitro maturation (IVM) of immature oocytes retrieved from unstimulated ovaries may avoid side effects connected to hyperstimulation during IVF procedures, including the risk of cancer recurrence. In humans, the scarce availability of immature oocytes limits morphological studies. The monovular ovine may represent an experimental model for IVM studies. To assess if the scarce developmental competence of prepubertal oocytes (PO) is related to morphological changes we analyzed, by light and transmission electron microscopy, cumulus-oocyte-complexes (COCs) from lambs (30-40 days old) and sheep (4-6 years old) at sampling and after 7 h, 19 h, 24 h of IVM. Meiotic progression was determined at the same time points. At sampling, the germinal vesicle (GV) of PO was round and centrally or slightly eccentrically located, whereas in adult oocytes (AO) it was irregularly shaped and flattened against the oolemma. PO, differently from AO, showed numerous trans-zonal projections. Organelles, including cortical granules (CGs), were more abundant in AO. After 7 h, the percentage of AO that underwent GVBD-MI transition increased significantly. In PO, the oolemma was juxtaposed to the ZP; in AO, it showed several spikes in correspondence of cumulus cells (CC) endings. In PO, organelles and isolated CGs were scattered in the ooplasm. In AO, groups of CGs were also present under the oolemma. After 19 h, PO underwent GVBD-MI transition; their oolemma showed several spikes, with CC projections retracted and detached from the ZP. AO underwent MI-MII transition; their oolemma regained a round shape. CGs were located beneath the plasmalemma, arranged in multiple, continuous layers, sometime discontinuous in PO. After 24 h, both groups reached the MII-stage, characterized by a regular oolemma and by expanded CCs. PO showed CGs distributed discontinuously beneath the oolemma, while AO showed a continuous monolayer of CGs. Even if PO were able of reaching morphological maturation after 24 h of IVM, our ultrastructural analysis allowed detecting the presumptive sequence of cytoplasmic alterations connected with the delay of nuclear maturation, that might explain the reduced developmental competence of such oocytes. Data from the sheep model are of interest for zootechny, and provide an experimental basis for improving human IVM technology.


Tissue segmentation-based electron density mapping for MR-only radiotherapy treatment planning of brain using conventional T1-weighted MR images.
Magnetic resonance imaging (MRI) is the primary modality for targeting brain tumors in radiotherapy treatment planning (RTP). MRI is not directly used for dose calculation since image voxel intensities of MRI are not associated with EDs of tissues as those of computed tomography (CT). The purpose of the present study is to develop and evaluate a tissue segmentation-based method to generate a synthetic-CT (sCT) by mapping EDs to corresponding tissues using only T1-weighted MR images for MR-only RTP. Air regions were contoured in several slices. Then, air, bone, brain, cerebrospinal fluid (CSF), and other soft tissues were automatically segmented with an in-house algorithm based on edge detection and anatomical information and relative intensity distribution. The intensities of voxels in each segmented tissue were mapped into their CT number range to generate a sCT. Twenty-five stereotactic radiosurgery and stereotactic ablative radiotherapy patients' T1-weighted MRI and coregistered CT images from two centers were retrospectively evaluated. The CT was used as ground truth. Distances between bone contours of the external skull of sCT and CT were measured. The mean error (ME) and mean absolute error (MAE) of electron density represented by standardized CT number was calculated in HU. The average distance between the contour of the external skull in sCT and the contour in coregistered CT is 1.0 ± 0.2 mm (mean ± 1SD). The ME and MAE differences for air, soft tissue and whole body voxels within external body contours are -4 HU/24 HU, 2 HU/26 HU, and -2 HU/125 HU, respectively. A MR-sCT generation technique was developed based on tissue segmentation and voxel-based tissue ED mapping. The generated sCT is comparable to real CT in terms of anatomical position of tissues and similarity to the ED assignment. This method provides a feasible method to generate sCT for MR-only radiotherapy treatment planning.


Ultrastructure of bone and cartilage formed in vivo in diffusion chambers.
Osteogenic tissue formed in vivo in diffusion chambers inoculated with a suspension of marrow cells contains both bone and cartilage. Observations by transmission electron microscopy on the transitional zone between these tissues showed a mixture of osseous and cartilaginous features in both matrix and cells. Two forms of collagen, morphologically consistent with Types I and II, are found in intimate association within the same intercellular septa. The results suggest the possibility that the different collagen types are synthesized by the same cells and that the variation in cellular morphology could be associated with the changes in the type of collagen synthesized. This unique characteristic of calcified tissues formed in diffusion chambers is probably the result of isolation from direct blood circulation, mechanical stress, and cellular mechanisms of tissue breakdown.


Methylenetetrahydrofolate reductase C677T genotype affects promoter methylation of tumor-specific genes in sporadic colorectal cancer through an interaction with folate/vitamin B12 status.
To evaluate joint effects of Methylentetra-hydrofolate reductase (MTHFR) C677T genotypes, and serum folate/vitamin B(12) concentrations on promoter methylation of tumor-associated genes among Iranian colorectal cancer patients. We examined the associations between MTHFR C677T genotype, and promoter methylation of P16, hMLH1, and hMSH2 tumor-related genes among 151 sporadic colorectal cancer patients. The promoter methylation of tumor-related genes was determined by methylation-specific PCR. Eighty six patients from whom fresh tumor samples were obtained and 81 controls were also examined for serum folate and vitamin B(12) concentrations by a commercial radioimmunoassay kit. We found 29.1% of cases had tumors with at least one methylated gene promoter. In case-case comparison, we did not find a significant association between methylation in tumors and any single genotype. However, in comparison to controls with the CC genotype, an increased risk of tumor methylation was associated with the CT genotype (OR = 2.5; 95% CI, 1.1-5.6). In case-case comparisons, folate/vitamin B(12) levels were positively associated with tumor methylation. Adjusted odds ratios for tumor methylation in cases with high (above median) versus low (below median) serum folate/vitamin B(12) levels were 4.9 (95% CI, 1.4-17.7), and 3.9 (95% CI, 1.1-13.9), respectively. The frequency of methylated tumors was significantly higher in high methyl donor than low methyl donor group, especially in those with MTHFR CT (P = 0.01), and CT/TT (P = 0.002) genotypes, but not in those with the CC genotype (P = 1.0). We conclude that high concentrations of serum folate/vitamin B(12) levels are associated with the risk of promoter methylation in tumor-specific genes, and this relationship is modified by MTHFR C677T genotypes.


Intracisternal delivery of NFκB-inducible scAAV2/9 reveals locoregional neuroinflammation induced by systemic kainic acid treatment.
We have previously demonstrated disease-dependent gene delivery in the brain using an AAV vector responding to NFκB activation as a probe for inflammatory responses. This vector, injected focally in the parenchyma prior to a systemic kainic acid (KA) injection mediated inducible transgene expression in the hippocampus but not in the cerebellum, regions, respectively, known to be affected or not by the pathology. However, such a focal approach relies on previous knowledge of the model parameters and does not allow to predict the whole brain response to the disease. Global brain gene delivery would allow to predict the regional distribution of the pathology as well as to deliver therapeutic factors in all affected brain regions. We show that self-complementary AAV2/9 (scAAV2/9) delivery in the adult rat cisterna magna allows a widespread but not homogenous transduction of the brain. Indeed, superficial regions, i.e., cortex, hippocampus, and cerebellum were more efficiently transduced than deeper regions, such as striatum, and substantia nigra. These data suggest that viral particles penetration from the cerebrospinal fluid (CSF) into the brain is a limiting factor. Interestingly, AAV2/9-2YF a rationally designed capsid mutant (affecting surface tyrosines) increased gene transfer efficiency approximately fivefold. Neurons, astrocytes, and oligodendrocytes, but not microglia, were transduced in varying proportions depending on the brain region and the type of capsid. Finally, after a single intracisternal injection of scAAV2/9-2YF using the NFκB-inducible promoter, KA treatment induced transgene expression in the hippocampus and cortex but not in the cerebellum, corresponding to the expression of the CD11b marker of microglial activation. These data support the use of disease-inducible vectors administered in the cisterna magna as a tool to characterize the brain pathology in systemic drug-induced or transgenic disease models. However, further improvements are required to enhance viral particles penetration into the brain.


Extended X-ray absorption fine structure investigation of adsorption and separation phenomena of metal ions in organic resin.
Analytical technique using organic resins has already been well-developed, and its applications are employed in various fields; nevertheless, the chemical phenomena occurring inside the resin remain unclear for the most part. In the present study, we apply EXAFS spectroscopy to elucidate the adsorption and separation phenomena of metal ions by organic resin. That is, the chemical species of trivalent lanthanides (Ln(III)) adsorbed in a tertiary pyridine resin from hydrochloric acid and nitric acid solutions have been determined by EXAFS. The results in HCl solutions suggest that Ln(III) ions are partly dehydrated in the resin phase, enabling the pyridine groups of the resin and chloride ions to coordinate to the Ln(III) ions in their primary coordination sphere. On the other hand, Ln(III) ions are tightly coordinated by several nitrate ions in HNO3 solutions and they keep forming the nitrate complex even in the resin phase. The lighter Ln of Nd tends to form an anionic nitrate complex, [Nd(NO3)4.nH2O]-, in the resin phase, while the middle Ln of Sm exists as a cationic nitrate complex, [Sm(NO3)2.nH2O]+, for the most part. On the basis of these EXAFS results, the adsorption and separation mechanisms of the pyridine resin in HCl solutions are interpreted as the direct coordination of pyridine groups to metal ions, while the mechanisms in HNO3 solutions are mainly dominated by the anion-exchange reaction between the protonated pyridine groups and the anionic nitrate complexes of Ln(III). The obtained results demonstrate that the hydration of metal ions weakens, and instead, other complexations are enhanced in the resin phase.