Fluorescein angiography has been so far the gold-standard test to assess diabetic macular ischemia (DMI), a cause of irreversible visual impairment in diabetic patients. The aim of this study was to investigate foveal avascular zone (FAZ) and perifoveal microcirculation changes in eyes with nonproliferative diabetic retinopathy (NPDR) using optical coherence tomography angiography (OCTA), a new and noninvasive vascular imaging technique.Cross-sectional study including eyes of diabetic patients with NPDR.All patients underwent medical history, best-corrected visual acuity (BCVA) measurement, slit-lamp and fundus examination, multicolor imaging, SD-OCT, and swept-source OCT. OCTA was performed in order to assess macular superficial and deep capillary plexus, and swept-source OCT was performed to evaluate the central choroidal thickness.P < 0.0001), surface (P = 0.0036) and perimeter (P = 0.0001), and for superficial capillary plexus nonperfusion index (NPI) (P = 0.0009). Moreover, a significant correlation was found between NPI and BCVA (P = 0.007).Fifty-eight eyes of 35 patients with a mean age of 61.8 years (±12.1) with mean HbA1C level of 7.6% (±1.5) were included in this study. Among them, 19 eyes had mild NPDR, 24 eyes had moderate NPDR, and 15 eyes had severe NPDR. There was a significant progression between NPDR stages for FAZ grade (OCT angiography is a useful noninvasive tool to explore early phases of diabetic retinopathy, which are not routinely explore with fluorescein angiography and not precisely enough with color photographs. NPI and foveal avascular zone parameters are correlated with glycated hemoglobin in patients with NPDR. If confirmed by further studies, these results could represent a mean to sensibilize diabetic patients to their disease. Visual impairment in diabetes is usually related to 3 different mechanisms: diabetic macular edema, complications of retinal neovascularization , and diabetic macular ischemia (DMI).Diabetes mellitus is a widespread condition affecting 366 million people worldwide. This pathological condition is characterized by enlargement of the physiological capillary-free zone located at the center of the macula, also known as the foveal avascular zone (FAZ), along with perifoveal capillary dropout.DMI has been reported to affect approximately 7% of patients with diabetic retinopathy.–7Various methods have been used to assess FAZ and perifoveal microcirculation: in vitro techniques on enucleated eyes as well as in vivo techniques, with psychophysical methods, fundus photography, and fluorescein angiography.–10The latter remains the gold-standard test, as it mainly explores the superficial capillary plexus which contains the perifoveal anastomotic capillary arcade defining the limits of the FAZ.Optical coherence tomography angiography (OCTA) is a newly available retinal vascular imaging technique, which is able to separately visualize superficial and deep macular capillary plexus.The aim of this study was to investigate foveal avascular zone and perifoveal microcirculation changes relative to mild, moderate, and severe nonproliferative diabetic retinopathy (NPDR) using the OCTA.22.1This cross-sectional study included diabetic patients affected with NPDR with appointments in the Department of Ophthalmology during July 2015. Patients were screened after informed consent was obtained. This study was performed according to the Declaration of Helsinki and was approved by the ethics committee of the French Society of Ophthalmology .2.2Inclusion criteria were age of 18 years or more and presence of an NPDR in the studied eye, as assessed on nonmydriatic fundus color photographs covering the Early Treatment Diabetic Retinopathy Study (ETDRS) 7-standard fields. For each patient, one or both eyes meeting those criteria were included.Noninclusion criteria were: presence of media opacities preventing reliable retinal imaging and fundus examination, macular edema with retinal thickness preventing good visualization of the FAZ by OCTA, other retinal diseases , history of vitreoretinal surgery, and history of macular laser grid.2.3All patients included in the study underwent comprehensive ophthalmologic examination including best-corrected visual acuity (BCVA) measurement on ETDRS scale, slit-lamp biomicroscopic and fundus examination, Spectral-Domain Optical Coherence Tomography , macular 30 degree MultiColor retinography  as well as ETDRS 35 degree 7-standard fields color retinal photographs  for retinal assessment, Swept-Source Optical Coherence Tomography  for choroidal thickness assessment, and Optical Coherence Tomography Angiography  for macular retinal vascularization assessment.Diabetic retinopathy was graded according to the International Diabetic Retinopathy Severity Scale, as previously described.Demographic data , general medical history , diabetic medical history , and ophthalmologic history  were also collected.2.4 This instrument uses a novel noninvasive and dyeless technique, which performs repeated sequential Optical Coherence Tomography cross-sectional scans at the same location on the retina in order to capture the dynamic motion of the erythrocytes. Thus, this device permits high-resolution imaging of the perfused vascular structure within specific layers of the retina with a 22-μm beam width. It has an A-scan rate of 70,000 scans per second, using a light source centered on 840 nm and a band-width of 45 nm. The tissue resolution is 5 μm axially, and the beam is 22-μm wide.For each included eye, an OCTA of the superficial and deep capillary plexus was performed as previously described.A 3 × 3 mm OCTA acquisition centered on the fovea was performed. The “en face” image was then automatically segmented with an inner boundary 3 μm beneath the internal limiting membrane and an outer boundary 15 μm beneath the inner plexiform layer (IPL) to obtain images of the superficial capillary plexus. The segmentation was carried out with an inner boundary 15 μm beneath the IPL and an outer boundary 70 μm beneath the IPL in order to obtain deep capillary plexus images.2.5 We manually traced FAZ contours in order to assess its different parameters. After setting the image scale to 3 × 3 mm in the software parameters, we were able to automatically obtain measurement of the perimeter and area of the FAZ.Each superficial capillary plexus image was analyzed using ImageJ 1.48 software .Similarly, the surface of the central nonperfused area visible on the deep capillary plexus image was measured.Furthermore, a nonperfusion index (NPI) of the total superficial capillary plexus image area was assessed using the same software by calculating the ratio between the cumulative area of pixels of which the brightness was beneath the value of 45 and the total area of the picture Fig. .2.6,15The extent of FAZ damage was also assessed using the ETDRS gradation system from 0  to 4 , as previously described.2.7P-value less than 0.20 from the univariate analysis, then a backward elimination procedure retained only independently significant variables. A logarithmic transformation was performed in order to normalize variable distribution if necessary. P < 0.05 was considered statistically significant. In this exploratory analysis, no correction was applied to account for intraclass correlation between eyes belonging to the same patient.Statistical analysis was performed with SAS software, version 9.3 . Qualitative variables are presented as number and percentage. Quantitative variables are presented as means and standard deviations. Nonparametric ANOVA comparing the 3 NPDR groups used the nonparametric Kruskal–Wallis test. Qualitative variables were compared by Fisher exact test. Univariate analysis of FAZ parameters was based on Spearman rank correlation coefficient when relating to quantitative variables and Mann–Whitney nonparametric test when relating to qualitative factors. Multivariate analysis used linear covariance analyses for each FAZ parameters. Covariates were entered in the analysis on a 33.1Fifty-eight eyes of 35 patients (mean age = 61.8 ± 12.1 years) with NPDR were included in this study  and 13 women (37%); type 2 diabetes affected 30 patients while 5 patients had type 1 diabetes. Mean diabetic duration was 18.3 ± 9.9 years, mean glycated hemoglobin A1C (HbA1C) level was 7.6 ± 1.5%, and 22 patients (63%) had oral medication and 29 patients (83%) had insulin therapy. Cardiovascular history of smoking, high blood pressure, and dyslipidemia were present in the majority of patients. These results are presented in Table 3.2Mean BCVA was 81.2 ± 12.3 letters (15–99). Only 1 eye had intravitreal antivascular endothelial growth factor history. Among the 58 included eyes, 19 eyes (32.8%) had mild NPDR, 24 eyes (41.4%) had moderate NPDR, and 15 eyes (25.8%) had severe NPDR (Table 3.3P = 0.034), oral diabetic medication (P = 0.002), and HbA1C (P = 0.049).Table 3.4P < 0.0001), FAZ surface (P = 0.0036), FAZ perimeter (P = 0.0001), superficial capillary plexus (SCP) NPI (P = 0.0009), and deep capillary plexus (DCP) central nonperfusion area surface (P = 0.0002). These results are reported in Fig. There was a statistical difference between the 3 NPDR groups for FAZ grade (3.5P = 0.033), between HbA1C and FAZ grade (P < 0.0001), surface (P = 0.0006), and perimeter (P = 0.0136). NPI was related to amputation history (P = 0.020), diabetes duration (P = 0.014), HbA1C (P = 0.039), and BCVA (P = 0.019). These results are indicated in Table Univariate analysis found significant associations between pseudophakia and FAZ surface  was observed between the NPDR groups, with a progressive increase of parameters according to NPDR stage. These results are not surprizing, as a significant increase in FAZ size is a well-known FA feature of diabetic retinopathy.Deep capillary plexus central nonperfused area was also significantly different between the 3 NPDR groups. However, OCTA image segmentation of the deep capillary plexus on OCT B-scan seemed relatively random and unreliable, and its clinical significance needs to be clarified.Finally, a statistical difference for NPI between the 3 NPDR groups was observed, with a progressive increase with NPDR stage. This is, to our knowledge, the first study to evaluate thoroughly and quantitatively macular microvascularization within a 3 × 3 mm square centered on the fovea in diabetic patients using an automated tool. Previous studies mostly used, besides FAZ measurements, a perifoveal intercapillary area index requiring manual marking of intercapillary spaces surrounding the fovea, with a different measuring protocol from one study to another. In addition to being automated, NPI has the advantage of taking into account, in a single standardized measurement, both FAZ surface enlargement and perifoveal capillary dropout. it did not appear in a more recent retrospective study conducted by Sim et al on FA images of a large cohort of patients, who nevertheless found a strong significant and independent association between papillomacular ischemia and BCVA. Thus, in our study, correlation between BCVA and NPI represents a new argument underlining the importance of perifoveal circulation in the occurrence of visual impairment secondary to DMI.Moreover, unlike FAZ parameters, in this study NPI was significantly associated with BCVA. In the literature, the correlation between BCVA and FAZ surface has not been consistently established. Although it was observed in a small series of 15 patients conducted by Arend et al, found a significant correlation between HbA1C and perifoveal intercapillary area, but not with FAZ surface. Conrath et al likewise failed to find evidence of a correlation between FAZ indexes  and HbA1C.In this study, HbA1C was positively correlated with FAZ surface, FAZ perimeter, FAZ grade, and NPI. In previous studies, however, associations between HbA1C and macular microcirculation parameters have been inconsistently described. In patients either without or with background retinopathy, Sander et alAnother positive correlation was observed between smoking status and FAZ grade and NPI, which is to our knowledge the first report of this association. In the context of diabetes, HbA1C and smoking correlated to the progression of FAZ and NPI indexes in NPDR patients could represent a mean to sensibilize this population.We acknowledge some limitations to this study. Indeed, OCTA was not compared to FA, the gold standard for FAZ evaluation. However, in NPDR assessment this comparison could be considered as questionable on account of ethical considerations. The sample size of this study was relatively small, but it was an exploratory analysis and sample size was determined by the statistician (PI).In conclusion, this study shows that OCTA is able to explore both superficial and deep macular capillary plexus in NPDR. With this noninvasive approach, progressive FAZ grade worsening and FAZ surface enlargement were observed during progression of NPDR. In addition, NPI, a newly described index, allows DMI assessment in a single automated and protocolized measurement taking into account both foveal and perifoveal microvascularization. Finally, even at early stages of diabetic retinopathy, HbA1C and smocking status seem to be associated with FAZ surface enlargement and NPI progression. If confirmed, these results could have therapeutic implications.The authors thank Jeffrey ARSHAM, an American medical translator, for rereading and reviewing our original English-language text. We are indebt to Optovue RTVue XR Avanti for providing us a device during the study.


The results shown here will provide basic information on APP-induced cytotoxicity at cellular and molecular levels.Atmospheric-pressure plasma (APP) has received attention due to its generation of various kinds of reactive oxygen/nitrogen species (ROS/RNS). The controllability, as well as the complexity, is one of the strong points of APP in various applications. For biological applications of this novel method, the cytotoxicity should be estimated at various levels. Herein, we suggest red blood cell (RBC) as a good cell model that is simpler than nucleated cells but much more complex than other lipid model systems. Air and N This means the reduction of fatty acid chain length by plasma treatment. There were more significant reductions at 1.59 ppm representing [−CO-CH2-CH2-], where the number of protons was measured as 53, 41, and 52 for control, N2 plasma-treated, and air plasma-treated RBCs, respectively. This implies the chemical changes of glycerol or fragmentation of phospholipids between fatty acid and glycerol. It is notable that N2 APPJ caused more severe effects on these changes.Though damages in cytoskeletal proteins, such as spectrin, actin, and band 3, could also lead to structural deformation , we firsreatment , 22.  Fi2 and air APPJ treatments, peaks at 3.26 and 3.65 ppm are significantly reduced, which represent 1H at the choline [−N(CH3)3] of phosphatidylcholine or sphingomyelin. In contrast, peaks at 3.16 ppm are significantly enhanced after only N2 APPJ treatments, which represents [−CH2NH3+] of phosphatidylethanolamine. Chemical reactions might possibly occur between the phospholipids and ammonium ions from N2 APPJ.Peaks between 3.73 and 5.22 ppm are generally related to the glycerol backbone, which connects the head and tail groups of the phospholipid. Most peaks remain after the plasma treatment; however, their relative ratio has changed. Peaks at 3.73 and 3.86 ppm are reduced by both plasma conditions, representing phosphatidylglycerol. However, peaks at 4.12 and 4.40 ppm are significantly reduced by only air plasma condition, representing glycerol Sn1 methylene protons. Sn1 carbon is connected to one part of acyl chains as Sn2 carbon, but it is more exposed to outer attacks, such as ROS from APPJ. In addition, a peak at 5.16 ppm newly appears in the case of air plasma treatment, which represents glycerol Sn2 protons connected to ether lipids. It is also notable that the peak at 5.37 ppm representing unsaturated fatty acid is almost conserved, and no significant peaks at 9.7 and 11 ppm representing aldehyde or carboxyl groups separately are observed.2 APPJ.In summary, our results imply that the outer head groups of the phospholipids or glycerols are vulnerable to the plasma treatment, as compared to the fatty acid tail groups inside the membrane, especially to air APPJ. The high power of removing the head group of phospholipids of air APPJ may result in reduction of the outer leaflet and following stomatocyte-like distortion. Likewise, the addition of ammonium ions on the head group may be the reason for echinocyte-like distortion of RBCs by N−1 corresponding to various pyrrole ring vibration modes and 1500–1640 cm−1 corresponding to the combination of spin state marker vibration modes. Peaks at 752 cm−1 and 798 cm−1 correspond to ring breathing, while peaks at 934 cm−1 and 674 cm−1 correspond to ring symmetric stretch and deformations of Hb. The protein bands (marked with “mem” in the figure) include mainly the amide I band at 1648 cm−1 and several low peaks of CH2/CH3 deformation modes primarily from amino acid side chains at 1448 cm−1, CH2 twist mode at 1324 cm−1, skeletal vibrations at 973 cm−1, and C-C stretch of phenylalanine ring at 998 cm−1. The lipid bands (marked with “mem” in the figure) include the acyl chain stretch modes at 2850–3000 and 1060–1180 cm−1 and CH2/CH3 deformation modes at 1448 cm−1.In order to obtain chemical changes in whole RBCs, we tried Raman spectroscopy with 488, 532, and 633 nm lasers. Different excitation wavelengths bring differential Raman spectra of RBCs, due to the specific absorption bands of RBCs . In prev2 APPJ treatment induced slight intensity changes, rather than frequency modulations. −1, and −1 became lower (corresponding to acyl chain stretch↓), the peak at 1370 cm−1 became sharper and higher (corresponding to pyrrol ring vibration: 1355 cm−1 representing Fe2+↓ and 1375 cm−1 representing Fe3+↑), and the wide band of multiple peaks at 1550–1650 cm−1  were modified by air APPJ treatment [−1 was an overtone of 1355 cm−1, whose intensities reduced together. N2 APPJ treatment reduced peaks at 1583 cm−1, which also represented the reduction of Fe2+-Hb.In the case of 488 nm laser , the peareatment . The pea−1↓), and iron of Hb may be oxidized to Fe3+ (1545 and 1601 cm−1 Fe2+↓ and 1637 cm−1 Fe3+↑) by air APPJ treatment. In addition, the sharp reduction at 752 cm−1 was related to the reduction of porphyrin breathing mode of Hb, which represents the viability of RBCs [−1 was related to the protein denaturation/heme aggregation, and that of the peak at 2430 cm−1 was related to the increase of hydrogen bonds of O-H…O type [−1 represented the reduced amount of CH2 or CH3 groups of acyl chains. Raman spectra of RBCs treated by N2 APPJ shows less difference to control, which means less impact on the lipid membrane and the Hb spin state by N2 APPJ.With 532 nm laser , the spe of RBCs . The enhH…O type , 27. The2 APPJ-treated RBCs showed the most difference from each other. As observed with 488 and 514 nm lasers, air APPJ treatment shortened the acyl chain length (1150–1170 cm−1↓), and reduced O2-bound Hb . Air APPJ treatment also enhanced the peaks at 1252 cm−1, which is rather strongly observed in met-Hb. N2 APPJ treatment also reduced the acyl chain length (1150–1170 cm−1↓). In addition, N2 APPJ treatment enhanced the peaks at 1549 and 1608 cm−1 and reduced the peaks at 977, 1238, 1392, and 1567 cm−1, which related to the reduction of O2-bound Hb [With 633 nm laser , the spebound Hb .2-bound Hbs to O2-detached form, or further to met-Hb (Fe3+ Hb without oxygen). Additionally, we analyzed them using PCA . This qualitative multivariate analysis helped us to discriminate one sample from the others. We performed PCA for RBC samples exposed to air APPJ, N2 APPJ, and H2O2. Four groups  seemed to be distinctly classified, but the boundary of control and N2 plasma-exposed RBC samples was found to be ambiguous. The result was the same when we tried the same analysis with lipid membrane ghosts from RBCs. These analyses suggest that air APPJ made distinct effects in comparison to N2 APPJ or H2O2, and the N2 APPJ made less change in the molecules of RBCs than air APPJ.Overall, Raman spectroscopy data show that both gas APPJs can affect the lipid membrane and even modify the spin states of intracellular Hb. It was difficult to discern peaks associated with proteins separately. APPJ reduced the length of acyl chains of lipid membrane, and reduced the O2 APPJ-treated RBCs are still red. The changes in absorption band denote the oxidation of Hbs in RBCs. Figures v or α band), and 575 nm (Q0 or β band) [3+ Hbs without oxygen (met-Hb), which are the oxidized form of Hbs by reactive species from air APPJ [2 APPJ or by 3 mM H2O2 are comparatively negligible  . Air APPair APPJ . In cont Figures . The enl Figures . Air APP2O2, cPTIO for NO·, SOD for O2∗-, and TA for OH· radicals. The spectra were displayed according to the increase of concentrations of each scavenger in the graphs. All scavengers alleviated the oxidation of RBCs; however, cPTIO and TA seemed more effective. The spectra remained at some level, even with high amount of CAT or SOD, which meant their reducing effects on Hb oxidation were limited to some levels. In contrast, the spectra recovered similar to control when cPTIO or TA was added, which implied NO∙ and OH∙ played major roles in Hb oxidations by air APPJ.In order to examine which reactive species were responsible for the met-Hb formation by air APPJ, we added several antioxidants to the RBC homogenates when they were exposed to air APPJ for 3 min. Figures 2 APPJ produced more amounts of OH∙ and H2O2, while air APPJ produced more amount of NO∙ in PBS solutions (2-bound Hb, resulting in met-Hbs and nitrate; reacts with Hb, resulting in nitrosyl-Hbs; and reacts with met-Hbs, resulting in Hb and nitric oxide cation [2O2 [The chemical analysis of APPJ-treated solutions showed that Nolutions . This ime cation , 31. Thue cation . In the ion [2O2 .Due to the high affinity of Hbs with NO∙, RBCs have regulatory roles in the modulation of intravascular NO∙. However, intravascular hemolysis releases Hbs into the plasma compartment, resulting in rapid rates of NO∙ consumption. Consequently, vascular relaxation and vasodilation are inhibited. It was reported that administration of blood Hb substitute solutions in clinical trials led to pulmonary and systemic hypertension, increased systemic vascular resistance, decreased organ perfusion and gastrointestinal paresis, and increased rates of death in trauma patients . APP tre2 APPJ have been shown to be able to induce morphological and hemolytic changes of RBCs in a time-dependent manner. Though N2 APPJ generates higher amounts of OH∙ and H2O2 oxidants, air APPJ elicits changes in RBCs at much shorter time through severe damage of the lipid membrane. In addition, air APPJ can alter intracellular O2-bound Hbs to met-Hbs, which loses O2 carrying potential. NO∙ generated in air APPJ may play critical roles in APPJ-induced cellular toxicity, including hemolysis, and iron-containing molecular oxidations. These results provide a useful biological model to estimate APPJ toxicity and provide insight into APPJ cytotoxicity in biological applications.In conclusion, both air and NFigure S1. Elasticity of APPJ-treated RBCs measured by atomic force microscopy. Figure S2. Raman spectroscopy of RBC and RBC lipid membrane after Air and N2 APPJ treatment and their PCA analysis.


OXTR) have been predicted to be involved in the regulation of social functioning in autism spectrum disorders (ASD). Objective of the study was to investigate serum OT levels and the OXTR rs2254298 polymorphism in Chinese Han children and adolescents with ASD as well as to identify their social deficits relevant to the oxytocinergic system. We tested serum OT levels using ELISA in 55 ASD subjects and 110 typically developing (TD) controls as well as genotyped the OXTR rs2254298 polymorphism using PCR-RFLP in 100 ASD subjects and 232 TD controls. Autistic symptoms were assessed by the Autism Behavior Checklist (ABC) and the Childhood Autism Rating Scale (CARS). There were no significant associations between OXTR rs2254298 polymorphism and ASD, serum OT levels and age, as well as serum OT levels and intelligent quotient (IQ) in both ASD and TD groups. However, ASD subjects exhibited elevated serum OT levels compared to TD controls and positive correlations between serum OT levels and “adaptation to change score” in the CARS and CARS total scores. Moreover, in the ASD group, significant relationships were revealed between the single-nucleotide polymorphism (SNP) rs2254298 and serum OT levels, the category “stereotypes and object use” in the ABC and the category “adaptation to change” in the CARS. These findings indicated that individuals with ASD may exhibit a dysregulation in OT on the basis of changes in OXTR gene expression as well as environmentally induced alterations of the oxytocinergic system to determine their social deficits.The neuropeptide oxytocin (OT) and its receptor ( Autism spectrum disorders (ASD) are a set of complex neurodevelopmental disorders characterized by deficits in social interaction and communication as well as restricted, repetitive behaviors  in the modulation of OT activities and the OT-OXTR signaling may be involved in ASD pathophysiology  in affiliation and social behavior in both animals and humans Carter, . Some stYamasue, , the OXTOXTR network. Centrally projecting neuropeptidergic neurons target regions that regulate stress coping, moods, and social behaviors  controls, several groups have reported lower plasma OT levels in individuals with ASD  in the third intron, rs2254298, has been identified as a particularly promising candidate to explain differences in oxytocinergic functioning , the ABC scores and the CARS scores. Moreover, the association between the OXTR rs2254298 polymorphism and ASD, as well as the relationships between OXTR SNP rs2254298 genotypes and serum OT levels, the ABC scores and the CARS scores were also analyzed. Finally, we hypothesized that Chinese Han children and adolescents with ASD may exhibit a dysregulation in OT on the basis of changes in OXTR gene expression as well as environmentally induced alterations of the oxytocinergic system to determine their social deficits.Although recent evidence supporting the essential role of OT and the OXTR rs2254298 polymorphism was genotyped by PCR-RFLP in 100 ASD subjects  and 232 age- and gender-matched TD controls  between 2 and 18 years of age. Participants for ELISA  were a subset of participants for PCR-RFLP . ASD subjects were diagnosed by qualified and experienced psychologists, psychiatrists or neurologists according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) criteria  and 110 age- and gender-matched TD controls  between 2 and 17 years of age. The The Autism Behavior Checklist (ABC) is a behavior checklist that consists of 57 items in 5 categories: “sensory”, “relating”, “stereotypes and object use”, “language”, and “social and self-help”. Each item corresponds to a single score referring to a single symptomatological area  is a 15-item behavior rating scale that includes 14 domains plus one category of general impressions that are rated on a four-point scale  according to interaction and observation. The 15 items are as follows: “relating to people”, “imitative behavior”, “emotional response”, “body use”, “object use”, “adaptation to change”, “visual response”, “listening response”, “perceptive response”, “fear or anxiety”, “verbal communication”, “non-verbal communication”, “activity level”, “level and consistency of intellective relations”, and “general impressions” . The tubes were gently inverted five times immediately and left undisturbed at room temperature for 30 min. Then, they were kept on ice until centrifuging at 1,731 × g for 10 min at 4°C. Serum was isolated and divided into 1-ml aliquots and immediately stored at −80°C until assay.DNA samples were obtained by an experienced technician by taking a sterile swab and rubbing it against the inside of participants' cheeks and placing it into a labeled sterile 2-ml Falcon tube.DNA was extracted from the swabs according to the manufacturer's protocol for the Swab Gen DNA Kit . The DNA solution for the current experiment was then stored at 4°C, and the redundant stock was stored at −20°C. The concentration of each DNA sample was determined using a NanoDrop ND-2000 spectrophotometer (Thermo Scientific).2O) was centrifuged at 17,000 × g for 15 min at 4°C. Supernatants were applied to the Sep-Pak C18 columns  and eluted off with a solution (acetonitrile/0.1% TFA-H2O ratio 60:40). Samples was evaporated at 4°C and stored at −20°C until reconstitution. Serum OT levels were measured using commercially available ELISA Kits , all steps in the manufacturer's instructions were followed, and results were read under 450 nm wavelength on a Power Wave-I microplate spectrophotometer . All assays were performed in duplicate. Standard curves and concentrations were computed by Excel. The assay range was from 2 pg/ml to 600 pg/ml. The intra-assay coefficient of variation was 6.9%, and the inter-assay coefficient of variation was 11.8%.Serum sample extraction procedures were previously carried out to decrease the potential cross-reacting molecules. A 1:1 mixture of serum sample and 0.1% trifluoroacetic acid in water (TFA-HOXTR SNP rs2254298 was amplified by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The primer sequences were as follows: 5′-TGA AAG CAG AGG TTG TGT GGA CAG G-3′ and 5′-AAC GCC CAC CCC AGT TTC TTC-3′ , 1.6 μl dNTP Mixture (2.5 mM), 1 μl each of the forward and reverse primers (10 μM/L), 3 μl genomic DNA, and 11.3 μl ddH2O. The PCR process included an initial denaturation step at 95°C for 5 min; 35 cycles of denaturation at 95°C for 30 s, annealing at 64.5°C for 30 s and extension at 72°C for 1 min; followed by a final extension for 7 min at 72°C. PCR products that were 307 bp long were visualized on a 2% agarose gel after electrophoresis. After the PCR products were digested at 65°C for 20 min with 0.5 μl of the restriction enzyme BsrI , the samples were run at 80 V for 90 min on an 8% polyacrylamide gel (acrylamide/bis-acrylamide ratio 19:1) and were visualized to distinguish the G allele  from the A allele . To ensure the accuracy of the RFLP profiles, gene sequencing was performed by Genewiz Inc, and the sequencing results agreed with the PCR-RFLP results.The 2 goodness-of-fit test. A χ2-test was also used in analyzing the genotype and allele frequencies, with a significance level of α = 0.05. A one-way analysis of covariance (ANCOVA) with sex, age, and IQ as covariates was applied for comparisons of ABC scores among three OXTR SNP rs2254298 genotypes. Kruskal-Wallis H tests were utilized for comparisons of serum OT levels and CARS total scores among three OXTR SNP rs2254298 genotypes. A ordinal polytomous logistic regression analysis was performed to discover the differences of the scores of each item in the CARS among three OXTR SNP rs2254298 genotypes . Odds ratios (ORs) were expressed with 95% confidence intervals (95% CI). Post-hoc Bonferroni correction for multiple comparisons was performed. A p < 0.05 was considered significant.The results were analyzed using SPSS17.0 software  and SAS version 9.3.2 . Kolmogorov-Smirnov test was performed to determine the normal distribution of the data. Levene's test was utilized to determine the homogeneity of variance. Differences in serum OT levels between two groups were examined with a Mann-Whitney U test and spearman rank-order correlations  were used to evaluate strength of associations. The Hardy-Weinberg equilibrium for the genotype frequency distributions of rs2254298 was assessed using a χZ = −2.178, p = 0.029). However, gender had no significant effect on serum OT levels in any comparison, between the two groups  and between males and females within the ASD group  and within the TD group  . No statistical differences were found from the genotypic and allelic frequencies of the OXTR SNP rs2254298 in ASD subjects and TD controls (p > 0.05) (Table The distribution of the 5) Table .H test detected differences among the three genotypes in the ASD group  , AA and GG genotypes (p = 0.095), as well as AG and GG genotypes (p = 1.000).Both blood and DNA samples were available for 55 ASD subjects and 110 TD controls. A Kruskal-Wallis r = 0.488, p = 0.000; Spearman partial correlation: r = 0.465, p = 0.001), suggestive of more social deficits with high serum OT levels. Moreover, serum OT levels also correlated positively with the “adaptation to change score” in the CARS , indicating that ASD subjects with higher serum OT levels were accompanied by worse abilities in adapting to novel environment. There were no significant corrections between serum OT levels and scores of other items in the CARS and ABC.ABC scores and CARS scores were only determined for ASD subjects. Spearman correlation and Spearman partial correlation  were both applied for all scores of the ABC and CARS. We found a significant positive correlation between serum OT levels and CARS total scores (Spearman correlation: p > 0.05) in two groups, including “ASD subjects for both OT and OXTR” (n = 55) and “ASD subjects for OXTR” (n = 100). In both groups, a one-way covariance (ANCOVA) with sex, age, and IQ as covariates indicated a significant relationship between genotypes and the “stereotypes and object use score” of the ABC . Further the Bonferroni post-hoc test revealed significant differences between the AA and AG genotypes in both ASD groups with different sample sizes  as well as between the AA and GG genotypes in “ASD subjects for OXTR” group  . Moreover, a ordinal polytomous logistic regression analysis with post-hoc Bonferroni correction was used to analyze differences of scores of each item in the CARS among OXTR SNP rs2254298 genotypes . Significant associations of “adaptation to change score” in the CARS among three genotypes were found in ASD subjects. ORs (95% CI) for “4 = severely abnormal” of “adaptation to change” among three OXTR SNP rs2254298 genotypes in “ASD subjects for both OT and OXTR” (n = 55) and “ASD subjects for OXTR” (n = 100) groups were shown in Table A Kruskal-Wallis H test followed by Bonferroni-corrected Mann-Whitney U test was applied to make the comparisons of CARS total scores among three genotypes of OXTR gene in social functioning. In the present study, we tested serum OT levels of both genders and elevated serum OT levels were observed in the ASD group compared to the TD group. Serum OT levels correlated positively with the “adaptation to change score” and “total scores” of the CARS. No associations were found between serum OT levels and age, as well as IQ in both ASD and TD groups. In addition, we made a comparison of the OXTR rs2254298 polymorphism in both groups and observed no statistical significance. However, significant associations were detected between OXTR SNP rs2254298 genotypes and serum OT levels, the “stereotypes and object use score” of the ABC and the “adaptation to change score” of the CARS in the ASD subjects.Animal and human studies, including studies of individuals with ASD, have provided evidence for the essential role of OT and the OXTR gene expression may also lead to elevated OT levels in ASD subjects. That is why we designed experiments on both serum OT levels and an OXTR gene polymorphism to find latent disturbances in ASD individuals.Our finding of higher serum OT levels in ASD participants was similar to several previous studies on plasma OT levels in individuals with ASD. For example, Jansen et al.  observedHowever, Miller et al.  reportedOverall, a substantial body of research has reported inconsistent outcomes in peripheral OT levels, which could be the result of differences among participants and investigative methods, including the multiple demographic characteristics of the ASD population , the blood sample collection time and the measurement and analysis of OT levels. Decreased OT levels in ASD might be a manifestation of the original genetic insult of ASD, and elevated OT levels in ASD might reflect a physiological response to ameliorate negative symptoms such as anxiety  was significantly associated with ASD, but SNP rs2254298 in the case-control test did not reveal any association with ASD except for the relationship with “daily living skills” and “communication” scores of the Vineland Adaptive Behavior Scales  revealed a significant genetic association with rs2254298A and rs53576A  group, and the severity of behavioral deficits in ASD participants with an AA genotype was significantly greater than the AG and GG genotypes in “ASD subjects for OXTR” (n = 100) group. Although no evidence for a direct association between serum OT levels and “stereotypes and object use score,” we found ASD subjects with AG and GG genotypes tend to have higher OT levels and lesser behavioral impairments (including restricted and repetitive behaviors). These results appeared to be consistent with the reduced repetitive behaviors after intranasal OT infusion .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nitrosocaldus clade, group 1.1b and group 1.1a Thaumarchaeota in terrestrial high-temperature habitats, only one§ enrichment culture of an AOA thriving above 50°C has been reported and functionally analyzed. In this study, we physiologically and genomically characterized a newly discovered thaumarchaeon from the deep-branching Nitrosocaldaceae family of which we have obtained a high (∼85%) enrichment from biofilm of an Icelandic hot spring (73°C). This AOA, which we provisionally refer to as “Candidatus Nitrosocaldus islandicus,” is an obligately thermophilic, aerobic chemolithoautotrophic ammonia oxidizer, which stoichiometrically converts ammonia to nitrite at temperatures between 50 and 70°C. “Ca. N. islandicus” encodes the expected repertoire of enzymes proposed to be required for archaeal ammonia oxidation, but unexpectedly lacks a nirK gene and also possesses no identifiable other enzyme for nitric oxide (NO) generation§. Nevertheless, ammonia oxidation by this AOA appears to be NO-dependent as “Ca. N. islandicus” is, like all other tested AOA, inhibited by the addition of an NO scavenger. Furthermore, comparative genomics revealed that “Ca. N. islandicus” has the potential for aromatic amino acid fermentation as its genome encodes an indolepyruvate oxidoreductase (iorAB) as well as a type 3b hydrogenase, which are not present in any other sequenced AOA. A further surprising genomic feature of this thermophilic ammonia oxidizer is the absence of DNA polymerase D genes§ – one of the predominant replicative DNA polymerases in all other ammonia-oxidizing Thaumarchaeota. Collectively, our findings suggest that metabolic versatility and DNA replication might differ substantially between obligately thermophilic and other AOA.Ammonia-oxidizing archaea (AOA) within the phylum Thaumarchaeota are the only known aerobic ammonia oxidizers in geothermal environments. Although molecular data indicate the presence of phylogenetically diverse AOA from the  In aquafestyles . Thaumarfestyles . In addion rates  and tranon rates  support § thermophilic AOA species from an enrichment culture has been reported to date (Candidatus (Ca.) Nitrosocaldus yellowstonensis.” The authors note that throughout this study the definition for thermophiles by Despite their apparent importance for nitrogen cycling in a wide range of thermal habitats, only one to date  and was amoA) gene trees “Ca. Nitrosocaldus yellowstonensis” branches most deeply among Thaumarchaeota that possess ammonia monooxygenase (AMO) genes. In consequence, the Nitrosocaldales clade has been considered as being close to the evolutionary origin of Thaumarchaeota encoding the genetic repertoire for ammonia oxidation  metabolic features of an obligately thermophilic AOA from the Nitrosocaldales clade obtained from a biofilm collected from a 73°C hot spring in Iceland. This organism, provisionally referred to as “Ca. N. islandicus” was initiated by inoculation of 40 ml sterile mineral medium  Iceland. At the sampling site, the spring had a pH of 6.5 and a temperature of 73°C. The culture was initially incubated without agitation in 100 ml glass bottles in the dark at 60°C and checked weekly for ammonium and nitrite content of the medium by using Nessler’s reagent  and nitrite/nitrate test stripes . Ammonium (1 mM NH4Cl) was replenished when completely consumed. At the same time pH was monitored by using pH test stripes (Macherey-Nagel) and kept at pH 7–8 by titration with NaHCO3. When the pH dropped below 6 the enrichment culture ceased to oxidize ammonia, but activity was restored by readjusting the pH to between 7 and 8. The ammonium and nitrite concentrations were quantified photometrically (in situ hybridization (FISH) with 16S rRNA-targeted probes labeled with dyes Cy3, Cy5, or Fluos as described elsewhere , which partly retains its antibacterial activity at 60°C (-5 to 10-8 to obtain a highly enriched (∼85%) AOA culture that was used for further characterization.The enrichment of “l medium  containitrically  using anlsewhere . Probes lsewhere , most arlsewhere  and mostlsewhere  were applsewhere  labeled  at 60°C , were peCa. N. islandicus” were determined across a range of incubation temperatures (50–70°C). Triplicate cultures (25 ml) and negative controls (cultures not supplied with ammonium or inoculated with autoclaved biomass) were incubated for 10 days in 100 ml Schott bottles without agitation in the dark at the respective temperature. Samples from these experiments were either stored at -20°C for subsequent qPCR analyses (150 μl) or centrifuged  to remove cells and the supernatant was stored at -20°C for chemical analysis (600 μl). qPCR analysis with primers CrenamoA19F . The specific growth rate was calculated from log-linear plots of amoA gene abundance in individual cultures. In this analysis, three out of seven time points were interpolated through linear regression.Growth rates of “namoA19F  and CrennamoA19F  targetinCa. N. islandicus,” 40 ml aliquots of mineral medium containing 1 mM ammonium were inoculated with 10% (v/v) of an exponential-phase culture and incubated in duplicates in the presence of 0, 33, and 100 μM PTIO, respectively. PTIO was dissolved in sterile mineral medium before addition to the cultures. The cultures not exposed to PTIO were supplemented with the same volume of sterile medium. The cultures were sampled (2 ml) at the beginning of the experiment and after 15 days of incubation. Nitrite and ammonium concentrations were measured as described above.To test whether the NO-scavenger 2-phenyl-4,4,5,5,-tetramethylimidazoline-3-oxide-1-oxyl  inhibits ammonia oxidation by “Ca. N. islandicus” as the only detectable ammonia oxidizer was extracted as described by DNA from three replicate enrichment cultures containing “Ca. N. islandicus” was uploaded to the MicroScope platform (Ca. N. islandicus” has been deposited in GenBank (accession CP024014) and associated annotations are publicly available in MicroScope . The raw sequence (Illumina MiSeq and Nanopore) data has been deposited to ENA under Study accession number PRJEB24462.The complete genome of “platform  for autoCa. N. islandicus” that lacked any homologs in other Thaumarchaeota were considered “unique.” Unique CDS of unknown function were searched for secretion signals and for predicted membrane-spanning domains of the encoded proteins using the Phobius web server , Neighbour Joining (NJ) and Maximum Parsimony (MP) methods. For all calculations, a sequence filter considering only positions conserved in ≥50% of all thaumarchaeotal and aigarchaeal sequences was used, resulting in 2444 and 488 alignment positions for the 16S rRNA and amoA genes, respectively.For 16S rRNA and  package  togetherA Bayesian-inference phylogenomic tree was obtained using the automatically generated alignment of 34 concatenated universal marker genes ]. Additional thaumarchaeotal polB sequences were identified using “Ca. N. islandicus” as a query in a blastp search against the nr protein database. These additional thaumarchaeotal sequences, the polB sequence from “Ca. N. islandicus” and arCOG database sequences were de-replicated using usearch ( usearch  with -so usearch  and a ph usearch .Nitrilase superfamily amino acid sequences were obtained from ior) sequences. Genomes available  from the NCBI genomes database were downloaded, genes were predicted using Prodigal V2.6.3 (iorA (TIGRFAM03336) using hmmsearch v3.1b22 with an e-value cutoff of 0.001. Genes meeting the search criteria were used as queries against the complete TIGRFAM database to ensure that the extracted iorA sequences matched the iorA model as the best-hit model with an e-value cutoff of 0.001. Reciprocal best-hit genes were required to align to the hmm over at least 500 contiguous bases. Amino acid sequences were then clustered into centroids using usearch v8.0.1517  was used to choose a small set of bacterial iorA sequences to include in the final tree. The final dataset was aligned using mafft v7.245  was based on l V2.6.3  and predt v7.245  and treet v7.245 .Ca. N. islandicus” cells were harvested by centrifugation  and fixed on poly-L-lysine coated slides with a filter-sterilized 2.5% glutaraldehyde fixation solution in phosphate buffered saline . Subsequently, fixed cells were washed three times for 10 min in PBS and post-fixed with a 1% OsO4 solution in PBS for 40 min. The fixed cells were again washed three times in PBS, dehydrated in a 30–100% (v/v) ethanol series, washed in acetone, and critical point dried with a CPD 300 unit (Leica). Samples were mounted on stubs, sputter coated with gold using a sputter coater JFC-2300HR (JEOL), and images were obtained with a JSM-IT300 scanning electron microscope (JEOL).For scanning electron microscopy, “Ca. N. islandicus” and accompanying bacteria over time as described in -1) followed by biomass transfers into fresh medium using serial dilutions. This enrichment culture showed near stoichiometric conversion of ammonium to nitrite when incubated at 65°C . This was accompanied by growth of the AOA with a specific growth rate of 0.128 ± 0.011 d-1 (mean generation time of 2.32 ± 0.24 d), which is substantially slower than those reported for “Ca. Nitrosocaldus yellowstonensis HL72,” Nitrososphaera viennensis EN76, or Nitrosopumilus maritimus SCM1 (Table 1), but faster than a marine enrichment culture , however, in a single experiment the optimal temperature was 70°C . The 16S rRNA gene and amoA gene of the newly enriched AOA are 96 and 85% identical, respectively, to the genes of “Ca. Nitrosocaldus yellowstonensis,” the only other cultured obligately thermophilic AOA. The average amino acid sequence identity (AAI) and the genomic average nucleotide identity (gANI) between the genome and the one of “Ca. N. yellowstonensis” are 65.4%  and 75.8% , respectively, which is above the proposed genus and below the proposed species boundary thresholds . An extended phylogenomic analysis using a concatenated alignment of 34 proteins . This result lends strong support to the earlier notion, which was based on single-gene 16S rRNA and amoA phylogenies . The enrichment level of “Ca. N. islandicus” was approximately 85% based on read counts from the Nanopore sequencing, whereas the Thermus sp. and Chloroflexi accounted for 12 and 3%, respectively. FISH-analysis of the enrichment culture confirmed the dominance of “Ca. N. islandicus” and showed that the AOA grew mainly in aggregates, whereas the bacterial cells grew either co-localized with the archaeal flocs or planktonically . Electron microscopy demonstrated that the cells of “Ca. N. islandicus” are small (with a diameter of approximately 0.5–0.7 μm) and have an irregular coccoid shape . Morphologically they resemble the cells of “Ca. N. yellowstonensis”  for sequences highly similar (97%) to the 16S rRNA genes of “Ca. N. islandicus” and “Ca. N. yellowstonensis” using the pipeline described by Ca. N. islandicus” and “Ca. N. yellowstonensis,” respectively) of the total microbial community . Interestingly, “Ca. N. yellowstonensis”-related organisms seem to occur mainly in hot springs described as alkaline with a pH of around 8.5, but were also detected in a sample from a Tibetan wastewater treatment plant  reduced the number of gene families identified as representing the “Thaumarchaeota-core”  cycle and for archaeal ammonia oxidation  is absent. NirK has been suggested to play an essential role for ammonia oxidation in AOA by providing NO for the NO-dependent dehydrogenation of hydroxylamine to nitrite , but lacks a cyanase that is used by Nitrososphaera gargensis for cyanate-based growth  and it will be interesting to find out for which metabolism they may be used in AOA.“ all AOA  . In this pathway arylpyruvates are formed from aromatic amino acids by the activity of amino acid aminotransferases using 2-oxoglutarate as the amine group acceptor. The glutamate produced by this transamination can be recycled back to 2-oxoglutarate via glutamate dehydrogenase (gdhA) with the concomitant reduction of NADP+. With ilvE and aspC genes present, “Ca. N. islandicus” encodes at least two enzymes for which an aminotransferase activity specific for tyrosine, phenylalanine and aspartate has been demonstrated . “Ca. N. islandicus” encodes four acetyl-CoA/acyl-CoA synthetases, two of which are most similar to non-syntenous homologs of acetyl-CoA/acyl-CoA synthetases found in other AOA. However, the third gene is absent in all other AOA to date and its most similar homologs are encoded by species of the peptidolysing thermophilic archaea Thermoproteus and Sulfolobus and the fourth is most similar to an acetyl-/acyl-CoA synthetase found in members of the thermophilic Bathyarchaea and Hadesarchaea.Intriguingly, “no acids  . However, no canonical ferredoxin:NADP+ oxidoreductase, or other enzymes  would be sufficient to compensate for all ferredoxin reduced in the dissimilatory fermentation pathway. However, “Ca. N. islandicus” can also oxidize reduced ferredoxin with a 2:oxoglutarate-ferredoxin oxidoreductase . NAD(P)H oxidation by this hydrogenase could lead to hydrogen generation, or the enzyme could act as a sulfhydrogenase that reduces zero valent sulfur compounds (produced by other organisms or present in the environment) to hydrogen sulfide (Ca. N. islandicus” to use hydrogen as energy source providing reduced NAD(P)H under oxic conditions as this type of hydrogenase has been shown to tolerate exposure to oxygen  and NADP enzymes  describe sulfide . The hyd sulfide . This hyo oxygen .Ca. N. islandicus” lacks genes for both subunits of the DNA polymerase D (PolD), which is present in all other AOA and most archaeal lineages (including thermophiles) with the exception of the Crenarchaea (Table 1). It is assumed that either PolD alone or together with DNA polymerases of the B family (PolB) is required for DNA synthesis and elongation in these archaea  does not encode the PolB1-binding proteins PBP1 and PBP2, which are required to form a multisubunit DNA polymerase holoenzyme together with PolB in the crenarchaeon S. solfataricus P2 , might help explain their sometimes surprisingly high abundance in anaerobic ecosystems (We have obtained a highly enriched (∼85%) culture of an obligately thermophilic AOA from a hot spring in Iceland. Despite the impressive diversity of AOA in high temperature environments as revealed by molecular tools , cultivad before . The newther AOA . Furtherosystems .Based on the data presented here, we propose the following provisional taxonomic assignment for the thaumarchaeon in our enrichment culture.Nitrosocaldales orderNitrosocaldaceae fam.Candidatus Nitrosocaldus islandicus’ sp. nov.‘Nitrosus (Latin masculine adjective): nitrous; caldus (Latin masculine adjective): hot; islandicus (Latin masculine genitive name): from Iceland. The name alludes to the physiology of the organism  and the habitat from which it was recovered.The biofilm of a terrestrial hot spring in Graendalur geothermal valley, Iceland .Ca. N. yellowstonensis”, are 65.4 and 75.8%, respectively.An obligately thermophilic, aerobic chemolithoautotrophic ammonia oxidizer from the phylum Thaumarchaeota growing as small irregular shaped cocci. The values of AAI and gANI between this species and its closest cultured relative, “Frontiers in Microbiology (doi: 10.3389/fmicb.2018.00028). The genomes of both organisms share many of the above discussed features such as the absence of nirK and genes encoding polymerase D, and thereby lend support to the possibility that these are common genomic features of the Nitrosocaldales.After submission of this manuscript a second report on the enrichment and genome analysis of a thermophilic thaumarchaeote was submitted and accepted for publication in Ca. N. yellowstonensis” genome. AD, JV, CS, PP, MW, and HD manually curated the annotation of the genome and interpreted the genome data. AD and MW wrote the manuscript with help from all co-authors.AD, JV, and CS cultivated and enriched the culture. AD, CS, and PP performed growth and activity experiments. AD performed FISH and SEM analysis. CH, AD, PP, MA, and RK performed bioinformatic analysis. JdlT kindly provided access to the “MA and RK own and run DNASense, the sequencing center at which the metagenomes were sequenced and the bins assembled. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The gossypol-MIP had higher selectivity (IF = 2.20) for gossypol over its structurally-similar analogs ellagic acid (IF = 1.13) and quercetin (IF = 1.20). The adsorption data of the MIP correlated well with the pseudo-second-order kinetic model and the Freundlich isotherm model, which implied that chemical adsorption dominated, and that multilayer adsorption occurred. Furthermore, the MIP exhibited an excellent regeneration performance, and the adsorption capacity of the MIP for gossypol only decreased by 6% after six reused cycles, indicating good application potential for selective adsorption of gossypol.The influence of various silica gel supports with different shapes and sizes on the recognition properties of surface molecular imprinted polymers (MIPs) was investigated. MIPs for selective recognition and adsorption of gossypol were synthesized via the sol–gel process with a surface imprinting technique on silica gel substrates. 3-aminopropyltriethoxysilane (APTES) and tetraethoxysilane (TEOS) were chosen as the functional monomer and the cross-linker. The morphology and structure of the gossypol-MIPs were characterized using Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and a standard Brunauer–Emett–Teller (BET) analysis. Results indicated that the surface imprinted polymer layer facilitated the removal and rebinding of the template, and thus, achieved fast binding kinetics. Compared with the MIPs prepared on irregularly shaped silica with a broad particle size distribution, the MIPs using regularly-shaped silica of uniform size showed higher imprinting factor (IF), and the MIP made with a relatively larger sized (60 μm) spherical silica, demonstrated higher adsorption capacity compared to the MIPs made with smaller sized, spherical silica. The MIP prepared with 60 μm spherically shaped silica, featured a fast adsorption kinetic of 10 min, and a saturated adsorption capacity of 204 mg·g The molecular imprinting technique is an attractive approach for molecular recognition with high affinity and selectivity ,2,3,4,5.Recently, molecularly imprinted sol–gel materials have also been reported ,23,24,25Cotton is one of the most important agricultural crops. The United States and China are two of the largest cotton producers worldwide. Gossypol is a toxic polyphenolic compound extracted from cotton plants ,28. Its For this purpose, various approaches have been developed, including ferrous sulfate treatment , solventAlong these lines, in the work reported here, MIPs for the selective recognition and adsorption of gossypol were designed by combining a sol–gel process with a surface imprinting technique on silica gel supports. Importantly, the influence of the shapes and sizes of silica gel supporting materials on the recognition properties of the surface gossypol imprinted polymers, were systematically investigated for the first time. The superiority of silica gel with a larger particle size and regular spherical shape, over other kinds of silica gel in preparing MIP, were then illustrated. The performance of the MIPs in the selective recognition and adsorption of gossypol, was evaluated using a solution adsorption experiment. The adsorption kinetics, adsorption capacity, adsorption isotherms, adsorption selectivity, as well as the reusability, were discussed in detail as well.Six different kinds of silica gel were chosen as supports to prepare the gossypol surface imprinted polymers. Silica spheres (0.5 μm average particle size) were obtained from Alfa Aesar ; silica spheres (5 μm average particle size) were obtained from Fuji Silysia Chemical Ltd. ; silica spheres (60 μm average particle size) were obtained from Acchrom Technologies Co., Ltd. ; irregularly shaped silica gel ,  and  were all purchased from Qingdao Haiyang Chemical Co., Ltd. . Gossypol was purchased from Sigma-Aldrich ; (3-aminopropyl)triethoxysilane (APTES) (98%) was purchased from Alfa Aesar ; tetraethoxysilane (TEOS) (98%), Quercetin (99%) and ellagic acid (98%) were purchased from Adamas ; methanol, acetone, acetic acid, hydrochloric acid, and sodium hydroxide were all analytical grade, and were purchased from Tianjin Zhiyuan Chemical Co., Ltd. . Water was purified using a Millipore Milli-Q gradient system  to high-performance liquid chromatography (HPLC) grade.A typical procedure for the synthesis of MIPs was as follows. Silica (8.0 g) and 60 mL of 33% (w/w) methanesulfonic acid aqueous solution were mixed in a 250 mL flask and refluxed for 8 h at 110 °C under stirring. After that, the resultant silica was washed with pure water to a neutral pH, and the silica particles were separated from solution by centrifugation. Then, they were dried under a vacuum at 70 °C for 10 h, and the activated silica particles were obtained .−1) were added to the mixture while stirring at room temperature for 24 h to obtain highly cross-linking polymer particles. The non-imprinted polymers (NIPs) were also prepared through the same procedure, without adding template gossypol [Gossypol  was first dissolved in 20 mL acetone. Then, APTES  was added, followed by stirring for 30 min to form monomer-template complex. After that, activated silica gel (400 mg) was added while stirring for 30 min to ensure silica particles were fully dispersed. Finally, TEOS  and acetic acid  until no template molecules were detected by a UV-Vis spectroscopy. Then, they were neutralized with 0.1 mol·L−1 NaOH solution and washed with ultra pure water and methanol. For NIPs, the particles were washed to a neutral pH with ultra pure water and methanol to remove the un-reacted substances. Finally, both MIPs and NIPs were dried at 80 °C for 12 h in a vacuum drying oven.To remove gossypol, the obtained polymer was repeatedly washed with 50 mL mixture of methanol and 6 mol·L−1 using KBr pellets. Nitrogen adsorption–desorption measurements were performed on an automatic adsorption instrument  and the specific surface areas (S) were calculated using the BET method.The binding capacity of the MIPs and NIPs were measured by UV-Vis spectroscopy . The morphologies of the samples were measured using field emission scanning electron microscopy (FESEM) . Fourier transform infrared (FT-IR) spectra were collected with a Bruker VERTEX-70 spectrometer  in the range of 4000–500 cmSolution binding studies were carried out to investigate adsorption performance of MIPs and NIPs for gossypol. In this study, the adsorption of gossypol was measured by UV-Vis spectrometry, and the typical UV absorption peak of gossypol at 373 nm was used for the quantitative analysis in the experiments.0, mg·L−1) of 110 mg·L−1, and were shaken at time intervals from 0 min to 60 min. Then, the supernatants and polymers were separated using syringe filters  and the concentrations of gossypol in the supernatant  were measured by UV-Vis spectrometry following a reported procedure [t, mg·g−1) at given times can be calculated according to Equation (1)In the kinetic adsorption experiments, 10 mg of MIPs or NIPs were added to 5.0 mL of gossypol solution in methanol at an initial concentration  using UV-Vis spectrometry [e, mg·g−1) was calculated according to Equation (2).In the adsorption isothermal experiments, 10 mg of MIPs or NIPs were added to 5 mL of gossypol solutions in methanol at various concentrations from 110 mg·Ltrometry . The equ−1 of gossypol, ellagic acid and quercetin solution in methanol. After being shaken for 1 h, the supernatants and polymers were separated, and the residual concentrations of gossypol, ellagic acid and quercetin were measured by UV-Vis spectrometry at 374, 366, and 371 nm, respectively. Additionally, the imprinting factor (IF) and selectivity coefficients (α) were used to evaluate the recognition and selectivity of MIPs and NIPs towards gossypol and competitive compounds. IF and α were calculated from Equations (3) and (4)MIP (mg·g−1) and QNIP (mg·g−1) represent the adsorption capacity of the templates, or analogs on MIPs and NIPs at the same conditions, respectively. IFT and IFC were the imprinting factors for gossypol and contrastive compounds, including ellagic acid and quercetin, respectively.In the adsorption selectivity experiments, two structurally-similar analogs of ellagic acid and quercetin were selected. 10 mg of MIPs or NIPs were added to 5 mL of each methanol solution of 200 mg·L−1 and was incubated for 1 h. After adsorption, the MIPs saturated with gossypol were collected by centrifugation, and were washed with a mixture of methanol and 6 mol·L−1 HCl  to remove gossypol completely, washed with ultra pure water to a neutral pH, and dried under vacuum. Then, the recovered MIPs were reused for rebinding of gossypol.The reusability of MIPs was investigated through six adsorption−desorption cycles. 10 mg of MIPs were added to 5.0 mL of gossypol solution in methanol at a concentration of 200 mg·L2 group, formed a complex through acid−base ionic pair interaction. APTES was not only the functional monomer, but also the intermediate to link silica particles [The schematic procedure for the synthesis of gossypol-MIPs was illustrated in articles . Then, t−1 and 1102 cm−1, indicating –OH and Si–O–Si vibration. The peaks around 800 cm−1 and 466 cm−1 were attributed to Si–OH and Si–O stretching vibrations [−1 and 1634 cm−1 represented N–H bond, and the 2933 cm−1 signal was associated to C–H bond. Moreover, it was found that there were no obvious differences between the spectra of the MIP and NIP. For the spectra of other samples in The FTIR spectra of the silica supports, and the corresponding NIPs and MIPs, were illustrated in brations , respectThe surface morphologies of the six activated silica gel particles and their MIPs and NIPs, are shown in 0 = 0.993, and the average pore diameter was obtained by Barret–Joyner–Halenda (BJH) method. 2·g−1, which was much smaller than all other five silica gels, including 5 μm spherical silica (280.9 m2·g−1), 60 μm spherical silica (431.4 m2·g−1), 10–40 μm irregular silica (299.9 m2·g−1), 50–75 μm irregular silica (275.9 m2·g−1) and 75–150 μm irregular silica (252.2 m2·g−1). The number of pores on the particle also affected the specific surface area. The more pores, the larger the specific surface area. The pore volume of 0.5 μm silica was 0.08242 cm3·g−1, and the pore volume of 5 μm silica was 0.8205 cm3·g−1, which was 10 times larger than 0.5 μm silica. The larger pore volume resulted from the particle having many more pores. Therefore, it was not difficult to understand that the specific surface area for the 0.5 μm silica was smaller than the other silica particles.MIPs were characterized with the nitrogen adsorption-desorption analysis. The specific surface area (S) was calculated using Multi-Point Brunauer–Emett–Teller (BET) methods, the total pore volume was estimated using a single point method at P/P2·g−1), there was a significant increase of surface area and pore volume for its corresponding MIP  and NIP . This could be due to the formation of the porous surface imprinting layer on silica support. Noticeably, for other larger silica support, there was a decrease in surface area and pore volume for the corresponding MIPs and NIPs. For example, the specific surface area and pore volume value of the 5 μm spherical silica  were higher than its MIP  and NIP . Furthermore, all the MIPs showed higher specific surface areas and pore volume than the corresponding NIPs. This implied that the imprinting process helped form more cavities and pores [2·g−1 and 0.3158 cm3·g−1, which were 1.5 times and 1.8 times of its corresponding NIP , respectively. The results were consistent with the fact that MIP normally showed larger surface area and pore volume than the non-imprinted material [In comparison to silica support, for example, 0.5 μm spherical silica  than the MIP (65.5 mg·g−1) made with spherical 0.5 μm silica, and MIP (51.4 mg·g−1) made with spherical 5 μm silica. Based on the imprinting factor and binding capacity data, 60 μm spherical silica was found to be superior support for preparing gossypol MIP and was selected for the subsequent experiments.The adsorption rate is an important parameter for the adsorption process. As shown in The adsorption performance of MIP prepared using 60 μm spherical silica was further studied in consideration of its outstanding adsorption capacity and excellent imprinting effect. e (mg·g−1) and Qt (mg·g−1) are the binding capacity of gossypol adsorbed at equilibrium and at time t (min), k1 (min−1) and k2 (g·mg−1·min−1) are the rate constant of pseudo-first-order and pseudo-second-order adsorption model, respectively.In order to probe the rate-determining step, pseudo-first-order (Equation (5)) and pseudo-second-order kinetic model ,46 (Equa2). It was found that the R2 values of the MIP and NIP by pseudo-second-order kinetic model, both exceeded 0.99, and the R2 value for MIP was as high as 0.9999. Moreover, the theoretical adsorption capacity  estimated from the pseudo-second-order kinetic model, perfectly agreed with the experimental adsorption data . The adsorption kinetic process perfectly fits the pseudo-second-order model, indicating that the chemisorption could be the rate-limiting step controlling the gossypol–binding process, and that the adsorption capacity was proportional to the number of active binding sites on the surface of the MIP [The parameters of adsorption kinetics were shown in  the MIP .−1. This indicated that the formation of the surface imprinted layer had high recognition ability and excellent imprinting effect for gossypol. The results also implied that the imprinted process formed the specific recognition sites for gossypol in the MIP matrix.To estimate the adsorption capability of the MIPs and NIPs, adsorption isotherm experiments were conducted with different initial concentrations of gossypol solution. e (mg·g−1) and Ce (mg·L−1) are adsorption capacity and free concentration of gossypol at adsorption equilibrium, respectively; Qm (mg·g−1) represents the maximum adsorption capacity of the adsorbent. KL (L·mg−1) is Langmuir constant, which is related to affinity of the binding sites. KF (mg·g−1) and n are both Freundlich constants that represent the adsorption capacity and adsorption favorability of the system, respectively. If n > 1, this suggests favorable adsorption.Langmuir (Equation (7)) and Freundlich (Equation (8)) isotherm models ,49 are w2 > 0.99). This implied that the binding sites in the MIP were heterogeneous, and the adsorption of gossypol onto the NIP could be partially monolayer adsorption.The nonlinear regression plots and the parameters fitted by two isotherm models are presented in In order to evaluate the selectivity of the MIP for gossypol, two structurally similar compounds, ellagic acid and quercetin, were chosen as control templates. As shown in As a promising gossypol adsorbent for future practical applications, regeneration potential and stability are vitally important. The used MIPs were recycled and reused for gossypol binding. As shown in Freundlich isotherm model was well fitted for the adsorption isotherm data of gossypol. The maximum binding capacity of the MIP was 204 mg·g−1, which was two times higher than that of NIP. The gossypol-MIP had higher selectivity for gossypol over its structurally similar analogues, ellagic acid and quercetin. Furthermore, the MIP demonstrated good stability and regeneration performance.In this study, a series of gossypol-MIPs were prepared by combining sol–gel strategy and surface molecular imprinting technique, using silica gel with different shapes and sizes as the supports. All the MIPs exhibited fast binding kinetics. Results indicated that the surface imprinted polymer layer facilitated the removal and rebinding of the template, and thus, achieved fast binding kinetics. Compared to the MIPs prepared on the silica of irregular shape and a broad particle size distribution, the MIPs using silica of regular spherical shape and uniform size, showed higher imprint factors. MIPs made with relatively larger sized (60 μm) spherical silica, demonstrated higher adsorption capacity than the MIPs made with smaller sized spherical silica. The MIP prepared on the 60-μm spherical silica reached equilibrium at 10 min, and its adsorption kinetics closely followed the pseudo-second-order kinetic model.


The use of D2/D3 dopaminergic agonists in Parkinson's disease (PD) may lead to pathological gambling. In a placebo-controlled double-blind study in healthy volunteers, we observed riskier choices in a lottery task after administration of the D3 receptor-preferring agonist pramipexole thus mimicking risk-taking behavior in PD. Moreover, we demonstrate decreased activation in the rostral basal ganglia and midbrain, key structures of the reward system, following unexpected high gains and therefore propose that pathological gambling in PD results from the need to seek higher rewards to overcome the blunted response in this system. The administration of dopaminergic drugs with D2/D3 agonist activity to Parkinson's patients, targeting the dopaminergic deficit in the nigrostriatal pathway in this condition, has recently been reported to lead to impulse control disorders, in particular pathological gambling, suggesting a breakdown of reward-hierarchies in such patients The underlying neural mechanism by which these dopaminergic agents modify the processing of rewarding stimuli and lead to compulsive gambling remains poorly understood. A recent fMRI study employing a gambling task found a decreased sensitivity of the reward system in pathological gamblers In order to test this hypothesis, we performed a slow event-related fMRI study in a group of 15 healthy young male volunteers using a 3 Tesla scanner. A double-blind, placebo-controlled cross-over design was used with placebo given in one session and 0.5 mg of pramipexole given 2 hours prior to the scan in the other. A lottery task was used in which participants could either win or lose 25 or 5 Euro cents in each trial see Fig. 1. previous outcomes and current choices. The analysis of the standard outcomes by means of a three-way ANOVA with treatment (placebo vs. pramipexole), type of previous outcome (gain vs. loss) and magnitude of outcome (25 or 5) showed a significant effect of type of previous outcome , with losses in the previous trial leading to riskier choices  than gains. Although, the overall number of times that subjects made a risky choice (25 cents) did not differ between treatments: 47% after placebo and 49% after pramipexole (t(12) = −1.0, p = 0.33); the analysis of the infrequent boost trials by means of a two-way ANOVA with treatment and type of boost trial (“+50” vs. “+10”) as factors showed a main effect of treatment . Pairwise comparisons by means of t-tests showed a significant effect for the “+50” trials only; t(12) = −2.28, p<0.05. Thus, pramipexole abolished the tendency to be conservative after a boosted win of 50 that was present under placebo. This behavioral pattern is consistent with clinical observations of an increase of risk behavior in Parkinson's patients treated with dopaminergic agonists.The behavioral data Fig. 2 sx,y,z: −15,−3,−3) in the globus pallidus. In the placebo condition, win trials  showed an increase in BOLD relative to loss trials (red). In particular the 50 cent boosted wins (blue thick line) gave rise to a pronounced increase of activity. By contrast, this increased response to unexpected high gains was absent in the pramipexole condition. Percentage bold increase to the boosted +50 condition under pramipexole was lower (0.58%) than after placebo (0.80%), t(12) = −2.44, p<0.05, potentially reflecting decreased phasic dopamine release Functional MRI results showed that monetary gains  in the placebo condition robustly increased activity in the rostral basal ganglia, in an area encompasing the globus pallidus and parts of the ventral striatum Fig. 3a.x,y,z:−4,−23,−8) was also assessed. Similarly to the results found in the rostral basal ganglia, the BOLD increase to the boost +50 trials (dark blue line) was lower under pramipexole (0.44%) than in the placebo condition (0.58%), t(12) = −2.24, p<0.05.Additionally, under placebo, unexpected high gains (boost +50) were associated with higher midbrain activity relative to normal gains  and +10 boost trials see Fig. 3b,While our main focus was on the responses in the ventral striatum and the midbrain, we also found significant activations in the contrast  in the anterior cingulate gyrus  and the posterior cingulate gyrus . All subjects gave their written informed consent and were paid for participation.The study was conducted according to a double-blind randomized crossover design. All volunteers received the two study medications, i.e., pramipexole and placebo, each subject acting thus as his own control. Volunteers participated in two different experimental sessions, separated at least by one week. On each experimental session participants received 20 mg domperidone in a non-blind fashion in order to antagonize any potential nausea induced by pramipexole. One hour later they received either placebo (lactose) or 0.5 pramipexole hydrochloride in a double blind fashion according to a randomization table. Half of the subjects received placebo in the first experimental session and pramipexole in the second. The other half received pramipexole in the first session and placebo in the second. The fMRI procedures were started two hours after pramipexole/placebo administration.The experimental paradigm consisted in a lottery task in which participants had to bet on one of two different numbers, 25 or 5, in order to increase a starting amount of 500 euro cents. Participants were instructed to choose one of the two numbers by button press. One second after the selection, the numbers changed color. Green indicated a win , red a loss . This feedback was shown for 2 s. The experimental session comprised two runs of 94 trials each. In 80 of the 94 trials, the feedback indicated a standard win or a standard loss. Additionally, in fourteen of the 94 trials, the so-called “boost trials”, wins were unexpectedly doubled and participants won 10 cents after choosing 5 (a green “10” was shown as feedback on the screen) and 50 cents after choosing 25 (a green “50” was shown as feedback on the screen). Thus, the six possible outcomes were: win 5, win 25, lose 5, lose 25, win 10 (“boost+10”) and win 50 (“boost+50”). No boost losses were programmed in the experimental paradigm. Participants were told that they had to adjust their choices in order to maximize their wins. In actual fact, the task was programmed to yield wins in 50% of the trials and losses in the other 50%. Participants were informed on the money they had won at three different times during each block. A slow event-related design was used with a constant interstimulus interval of 12 s, during which a fixation cross was presented in the center of the screen.Data were acquired in a 3-Tesla Siemens Magnetom Trio Scanner. First, structural images of the brain were obtained by means of a T1-weighted MPRAGE sequence: 256×256 matrix; field of view (FOV) = 256 mm; 192 1-mm sagittal slices. Subsequently, functional images were obtained in two runs implementing an echo-planar-imaging sequence. The pulse-sequence parameters were as follows: time to repeat (TR) = 2000 ms; time to echo (TE) = 30 ms; FOV = 224 mm; flip angle (FA) = 80°; matrix = 64×64; slice thickness = 4 mm. Thirty-two transversal slices (3.5×3.5×4 mm voxel) were obtained parallel to the anterior commissure-posterior commissure (AC-PC).Usable data were finally obtained from 13 volunteers. One volunteer withdrew from the study after complaining of visual distortions which disappeared when he was removed from the scanner. Another participant invariably chose “25” in 100% of the trials throughout the two experimental sessions, thus failing to show any attempt to modulate his behavior based on prior outcomes.Data analysis included preprocessing , co-registration and normalization to Talairach stereotaxic space using Brain Voyager QX. Random-effects analysis was performed for the functional data  including the following predictors: “win+25”, “win+5”, “loss-5”, “loss-25” and “boost+10” and “boost+50” for the placebo and pramipexole conditions. Predictors were convolved with a two-gamma hemodynamic response function with the following parameter values: onset = 0, time to peak = 5 s, time to undershoot peak = 15 s.Results were considered statistically significant when p values were lower than 0.001 uncorrected for multiple comparisons.The time course of the BOLD response was assessed by defining regions of interest ROIs around the voxel of maximum statistical difference. The ROIs defined extended 5 voxels in the x,y,z directions. For direct statistical comparisons of ROI activation in a given condition between treatments, the maximum percentage of increase was obtained for each participant and these values compared by means of pairwise t-tests. Results were considered statistically significant when p values were lower than 0.05.The probability of making “risky” choices, i.e., choosing 25, was measured in relation to the outcome on the previous trials. These values were analyzed by means of repeated measures ANOVAs. Factors included treatment , type of previous outcome  and magnitude of outcome . Results were considered statistically significant when p values were lower than 0.05.


In “The Changing Epidemiology of Malaria in Minnesota,” S.A. Seys and J.B. Bender, a number was inadvertently inserted into a sentence. On page 993, column 2, second full paragraph, the third sentence should read, “Among the patients who were born abroad, sites of malarial infection were Africa and Asia.” We regret any confusion this error may have caused.


Rickettsia honei strain “marmionii”), we describe 1 case of Queensland tick typhus  and 2 cases of scrub typhus caused by a unique strain (Orientia tsutsugamushi).We report 3 rickettsioses on Darnley Island, Australia, in the Torres Strait. In addition to previously described cases of Flinders Island spotted fever ( Darnley (Erub) Island is the largest island  (R. typhi), scrub typhus (Orientia tsutsugamushi) (R. honei strain “marmionii”)  sought treatment for fever (39.3°C), headache, and an eschar on his right thigh. He had no rash. No diagnosis was made, but he was given penicillin V. Seven days later he returned with worsening symptoms of fever (39.9°C), headache, cough, arthralgia, and lethargy. A provisional diagnosis of scrub typhus was made, and he was given doxycycline. By the next day he was afebrile, and his condition was much improved. The diagnosis was confirmed by a positive serologic result for scrub typhus rickettsiae (antibody titer 512) and the presence of  culture .O. tsutsugamushi (and it was later isolated) in his day-10 blood sample. Follow-up serologic testing of samples collected on days 10 and 33 displayed seroconversion to antibodies against scrub typhus .In May 2004, an 11-year-old boy (patient 2) was examined because of a 3-day history of fever (39.0°C), headache, nausea, abdominal pain, and a boil surrounded by cellulitis behind his left ear. He had no rash. He was given flucloxacillin for the boil. On day 10 he was still ill and febrile. His blood was tested for malaria, dengue, and scrub typhus, but he was given no antimicrobial therapy. He was not seen again until day 18, when he was prescribed doxycycline after PCR detected In April 2003, a 29-year-old man (patient 3) sought treatment for fever (38.4°C), cough, nausea, lethargy, and cellulitis of both feet. His left thigh had 2 boils, and his inguinal nodes were palpable. Three days later, he was evacuated by air and admitted to the Thursday Island hospital, at which time he was still febrile (39.2°C) but had no cellulitis. He also had a macular rash, which he reported as originally having been pustular; the thigh lesions were recognized as eschars. A tentative diagnosis of scrub typhus was made, and the patient was given doxycycline. Within 48 hours he was afebrile and discharged. Serologic testing for rickettsiae was positive for the spotted fever group (titer 256) on day 3, but the titers remained unchanged 4 months later. A spotted fever group rickettsial organism was grown from the day-3 blood sample; however, PCR was negative for rickettsiae .>128 were deemed positive. Patient 1 had an increase in titer to O. tsutsugamushi; patient 2 exhibited seroconversion to O. tsutsugamushi antibodies; and patient 3 had stationary positive titers to spotted fever group rickettsiae .Rickettsial isolation was performed according to previously described methods  and following the manufacturer’s protocols. Scrub typhus was diagnosed by 56-kDa gene PCR, which used the primers A (5′-TACATTAGCTGCAGGTATGACA-3′) and B (5′-CCAGCATAATTCTTTAACCAAG-3′)  as previously described, without the nested procedure and with a 51°C annealing temperature . The patient’s buffy coat DNA extract was not tested for PCR inhibitors, and no attempt was made to use the R. australis isolate in a heterologous serologic reaction because the strain could not be established in continuous culture.Spotted fever group rickettsemia was identified by 17-kDa antigen gene PCR that used the primers MTO-1 (5′-GCTCTTGCAACTCTATGTT-3′) and MTO-2 (5′-CATTGTTCGTCAGGTTGGCG-3′) (Invitrogen) as previously described, with an annealing temperature of 51°C and 45 cycles   to the spotted fever group in the scrub typhus patients  are consSince 1935, numerous cases of scrub typhus have been reported in Australia and Papua New Guinea . Hence,


Escherichia coli (STEC) provide significant epidemiological information on clinical isolates. Each O-antigen determinant is encoded by a unique cluster of genes present between the gnd and galF chromosomal genes. Alternatively, serogroup-specific polymorphisms might be encoded in loci that are encoded outside of the O-antigen gene cluster. Segments of the core bacterial loci mdh, gnd, gcl, ppk, metA, ftsZ, relA and metG for 30 O26 STEC strains have previously been sequenced, and comparative analyses to O157 distinguished these two serogroups. To screen these loci for serogroup-specific traits within a broader range of clinically significant serogroups, DNA sequences were obtained for 19 strains of 10 additional STEC serogroups. Unique alleles were observed at the gnd locus for each examined STEC serogroup, and this correlation persisted when comparative analyses were extended to 144 gnd sequences from 26 O-serogroups (comprising 42 O : H-serotypes). These included O157, O121, O103, O26, O5 : non-motile (NM), O145 : NM, O113 : H21, O111 : NM and O117 : H7 STEC; and furthermore, non-toxin encoding O157, O26, O55, O6 and O117 strains encoded distinct gnd alleles compared to STEC strains of the same serogroup. DNA sequencing of a 643 bp region of gnd was, therefore, sufficient to minimally determine the O-antigen of STEC through molecular means, and the location of gnd next to the O-antigen gene cluster offered additional support for the co-inheritance of these determinants. The gnd DNA sequence-based serogrouping method could improve the typing capabilities for STEC in clinical laboratories, and was used successfully to characterize O121 : H19, O26 : H11 and O177 : NM clinical isolates prior to serological confirmation during outbreak investigations.Serogroup classifications based upon the O-somatic antigen of Shiga toxin-producing  Escherichia coli (STEC) are bacterial pathogens that result in both outbreak and sporadic occurrences of human mortality and disease. Symptoms can include bloody and non-bloody diarrhoea, and children are susceptible to renal failure due to haemolytic uraemic syndrome. STEC are transmitted to humans by consumption of contaminated food or water, person-to-person contact or animal-to-person contact, where natural reservoirs include cattle, pigs and sheep  pathogenicity island, and the stx1 and stx2 loci encoding Shiga toxins  and JUMPstart sequence, and enzymic digestion of this amplicon identified RFLPs correlating to O-antigen determinants  and boiling the cells for 15 min. Boiled cells were pelleted, and the supernatant was removed and used as the DNA template in PCR.mdh, gnd, gcl, ppk, metA, ftsZ, relA and metG are presented in Table 2Taq (Invitrogen), following the manufacturer’s directions. The thermocycling parameters for ftsZ, relA and metG included an initial denaturation at 94 °C for 5 min, 35 cycles of denaturation at 94 °C for 40 s, annealing at 50 °C for 45 s and extension at 68 °C for 45 s, with a final extension at 68 °C for 5 min. The annealing temperature for metA, mdh, gcl and ppk was 58 °C, and 52 °C for gnd. PCR products were purified using the QIAquick PCR purification kit (Qiagen) and sequenced using the same primers that generated these amplicons. Sequencing was performed on an ABI3730 (Applied Biosystems) and the data were deposited in GenBank with accession nos DQ472524–DQ472651. Existing genomic sequence data for E. coli O157 : H7 EDL933, O157 : H7 Sakai, O6 : H1 CFT073 and K-12  was included in our dataset for each of the above loci. From directed studies against the gnd locus , neighbour-joining trees were constructed with Hasegawa–Kishino–Yano (HKY85) distance correction using SplitsTree4 .Multiple sequence alignments were completed using ClustalW  were selected for further study. This panel included further strains from the serotypes represented in Fig. 1E. coli strains recovered from paediatric stool samples . The overall genetic distinction between STEC serogroups (as determined in the eight locus scheme) was also represented amongst these four loci, and the additional strains and serogroups . The overall genetic distinction between serogroups (as determined in the eight and four loci schemes) was also represented in this single locus, as each examined STEC O-serogroup encoded a unique gnd allele , and for each serogroup all STEC strains encoded an identical gnd allele  also encode similar O-antigen gene clusters, nor are the antigens themselves similar. The potential utility of a locus subject to recombination between genera might be seemingly limited for the purpose of molecular-based serogrouping; however, we currently observed conserved STEC serogroup-specific genetic polymorphisms at the gnd locus. Between strains of an individual STEC O-serogroup we observed conserved gnd alleles, and no serogroup encoded a gnd allele that was identical to another serogroup. This study provides a simple method for molecular-based serogrouping of E. coli strains encoding stx, which can be detected by a wealth of molecular reagents (The proportion of synonymous and nonsynonymous mutations was calculated for each locus from the accumulated DNA sequence data (Table 3ore loci . A compalineages . To our


In total, 20 patients with locally advanced or metastatic NSCLC who received no prior chemotherapy or one previous chemotherapy regimen were enrolled. The most frequent toxicities were neutropenia and thrombocytopenia; nonhaematological toxicities were generally mild. Three out of six patients experienced dose-limiting toxicities  at dose level 4, 100 mg m−2 nedaplatin with 1000 mg m−2 gemcitabine, which was regarded as the MTD. There were three partial responses, for an overall response rate of 16.7%. The median survival time and 1-year survival rate were 9.1 months and 34.1%, respectively. This combination is well tolerated and active for advanced NSCLC. The recommended dose is 80 mg m−2 nedaplatin with 1000 mg m−2 gemcitabine. This combination chemotherapy warrants a phase II study and further evaluation in prospective randomised trials with cisplatin- or carboplatin-based combinations as first-line chemotherapy for advanced NSCLC.To establish the toxicities and maximum tolerated dose (MTD) of nedaplatin with gemcitabine, and to observe their antitumour activity, we conducted a combination phase I study in advanced non-small-cell lung cancer (NSCLC). Patients received nedaplatin (60–100 mg m Based on the results of a meta-analysis , cisplat−2, respectively, and the dose-limiting toxicity (DLT) was thrombocytopenia  ⩽25 mg dl−1, PaO2 ⩾60 mmHg or SpO2 ⩾90%]; and (5) normal electrocardiogram (ECG). At least 4 weeks must have passed after the completion of previous therapy and the patients had to have recovered from the toxic effects of previous therapy. The exclusion criteria consisted of pulmonary fibrosis or interstitial pneumonitis with symptoms or apparent abnormalities on chest X-ray, massive pleural effusion or ascites, acute inflammation, pregnancy, lactation, symptomatic brain metastases, active concurrent malignancies, severe drug allergies, severe heart disease, cerebrovascular disease, uncontrollable diabetes mellitus or hypertension, severe infection, active peptic ulcer, ileus, paralysis intestinal, diarrhoea and jaundice. This study was performed at Kinki University School of Medicine and was approved by the Institutional Review Board. Written informed consent was obtained from all patients. This study was conducted in accordance with Declaration of Helsinki.Patients with histologic or cytologic confirmation of locally advanced or metastatic NSCLC who received either no prior chemotherapy or one previous chemotherapy regimen were eligible. The eligibility criteria were as follows; (1) measurable lesions; (2) age ⩽75 years; (3) Eastern Cooperative Oncology Group (ECOG) performance status (PS) 0–1; (4) adequate organ function (a white blood count (WBC) ⩾4000 Prior to entry, a complete history was taken and physical examination including age, height, weight, performance status, histological diagnosis, tumour stage, contents of previous treatment and presence of a complication was performed. The pretreatment laboratory investigations included a complete blood cell count, differential WBC count, platelet count, serum electrolytes, total protein, albumin, total bilirubin, transaminase, alkaline phosphatase, lactate dehydrogenase, BUN, creatinine, creatinine clearance and urinalysis. After the initiation of therapy, a complete blood cell count with a differential WBC count was performed at least twice a week. Blood chemistry profiles and chest X-ray films were obtained weekly. The lesion measurements were performed during at least every second course. Toxicities were evaluated according to the National Cancer Institute Common Toxicity Criteria (NCI-CTC) version 2 and tumour responses were assessed using the Response Evaluation Criteria in Solid Tumors (RECIST) guidelines .μl−1 and/or the platelet count was >750 000 μl−1, and/or allergic reaction, fever, elevation of transaminase and pneumonitis were less than grade 2, and/or the other nonhaematological toxicities were less than grade 3. The subsequent courses were withheld until the toxic levels returned to those specified in the eligibility criteria. The doses of both drugs were decreased by one dose level if DLTs occurred. In the case of the initial dose level, the doses of nedaplatin and gemcitabine were reduced by 20 and 200 mg m−2, respectively.The treatment schedule included nedaplatin, diluted with 500 ml of normal saline, given intravenously over 90 min on day 1, and gemcitabine with 100 ml of normal saline, given intravenously over 30 min after the completion of nedaplatin infusion on days 1 and 8, every 3 weeks. All patients were allowed to receive antiemetics with dexamethasone and granisetron, and post-therapy hydration with 1000 ml of normal saline. Granulocyte colony-stimulating factor (G-CSF) prophylaxis was not administered. Doses of gemcitabine on day 8 were given if the WBC count was >2000 μl−1, (4) grade 3 febrile neutropenia, (5) grade 3 nonhaematologic toxicity except for nausea/vomiting, (6) delay of administration of gemcitabine on day 8 over a week for toxicities.Dose escalations were performed as listed in Between August 2001 and February 2003, 20 patients were enrolled in this study. The total and the median number of courses were 56 and 3 (range 1–6), respectively. The patients’ characteristics are shown in The haematological and nonhaematological toxicities observed during the first course are shown in μl−1. The third patient could not receive the second course due to the delayed anaemia, also considered as DLT. Therefore, dose level 4, 100 mg m−2 nedaplatin with 1000 mg m−2 gemcitabine was regarded as the MTD. The recommended dose level for further phase II study was determined to be 80 mg m−2 nedaplatin with 1000 mg m−2 gemcitabine (dose level 3 in this study).At levels 1 and 2, none of the patients had developed a DLT. Haematological and nonhaematological toxicities were generally mild at these levels, although one patient had grade 3 thrombocytopenia at level 1. At level 3, two of six assessable patients had developed DLTs. Both could not receive their scheduled dose of gemcitabine on day 8 because they had neutropenia, thrombocytopenia and high transaminase activity. At level 4, three of six patients had developed DLTs. One patient received G-CSF for neutropenia, not lasting more than 4 days, which was considered as the DLT. Another patient required a platelet infusion because of thrombocytopenia <20 000 There were three partial responses, for an overall response rate of 16.7%. As for squamous cell carcinoma, only one out of seven patients had a partial response. The median progression-free survival time was 5.1 months. The median survival time and 1-year survival rate were 9.1 months and 34.1%, respectively. Out of 15 patients who had received prior treatment, two (13.3%) achieved a partial response, and there was no clear relationship between responses to previous treatment and responses to this regimen. For previously treated patients, the median survival time and 1-year survival rate were 9.2 months and 40.3%, respectively. Among five previously untreated patients, one (20%) achieved a partial response and the median survival time and 1-year survival rate were 12.0 months and 50.0%, respectively.Many recent randomised clinical trials have shown that the combinations of cisplatin with one of the new agents, such as gemcitabine, taxanes or vinorelbine, is the standard therapy for patients with locally advanced or metastatic NSCLC . Howeverin vivo study suggested that nedaplatin–gemcitabine resulted in more enhanced inhibition of tumour growth than cisplatin–gemcitabine or carboplatin–gemcitabine. These results prompted us to investigate nedaplatin-based combinations and to conduct this phase I study.Like carboplatin, nedaplatin is also a second-generation platinum derivative that appears to have a similar mechanism and toxicity profile to carboplatin, although direct comparison has not been performed. Moreover, With respect to toxicities, the most frequent toxicities were haematological toxicities, especially neutropenia and thrombocytopenia. Eight of 18 patients (44.4%) developed more than grade 3 neutropenia after the first courses, and after 16 out of 56 (28.6%) courses overall. On the other hand, six out of 16 patients (37.5%) developed grade 3 thrombocytopenia after the first courses, and after 16 out of 56 courses (37.5%) overall. However, patients required platelet transfusions during only three courses. In addition, one previously untreated patient developed drug-related pneumonitis, which improved with the administration of steroid, at level 3 after the fifth course.Overall, the toxicities of the combination of nedaplatin with gemcitabine were generally mild and this combination chemotherapy is both well tolerated and active against advanced NSCLC.The overall response rate of 16.7%, the median survival time of 9.1 months, and 1-year survival rate of 34.1% in this study were quite acceptable because most patients had been given prior chemotherapy. As evaluation of antitumour activity was not a primary objective, and our patient population was small and heterogeneous, we are unable to draw definitive conclusions about the activity of this regimen. Currently, it is still controversial whether novel platinum compounds such as carboplatin and nedaplatin could replace cisplatin for the treatment of advanced NSCLC. However, when not only antitumour activity but also palliation are the main goals of treatment, these new platinum compounds might play a useful role because of their favourable toxicity profile. Therefore, nedaplatin–gemcitabine warrants a phase II study, and further evaluation in prospective randomised trials with cisplatin- or carboplatin-based combinations as a first-line chemotherapy for advanced NSCLC in order to investigate whether nedaplatin could replace cisplatin or carboplatin.−2 nedaplatin with 1000 mg m−2 gemcitabine and 80 mg m−2 nedaplatin with 1000 mg m−2 gemcitabine, respectively.In conclusion, the combination of nedaplatin with gemcitabine is well tolerated and active for advanced NSCLC. The MTD and recommended dose level are 100 mg m