Peri-urban farmland provides a diversity of ecological services. However, it is experiencing increasing pressures from urban sprawl. While the effects of land use associated with farming on arthropod assemblages has received increasing attention, most of this research has been conducted by comparing conventional and organic cropping systems. The present study identifies the effects of urban sprawl and the role of non-cropped habitat in defining arthropod diversity in peri-urban farmed landscapes. Multi-scale arthropod data from 30 sampling plots were used with linear-mixed models to elucidate the effects of distance from urban areas  on arthropods. Results showed that urban sprawl, disturbed farm landscapes, and disturbance in non-cropped habitats had negative effects on arthropods, the latter requiring arthropods to re-establish annually from surrounding landscapes via dispersal. While arthropod species richness showed no obvious changes, arthropod abundance was lowest in zone II. Generally, patch density (PD), Shannon diversity index (SHDI), and aggregate index (AI) of non-cropped habitat were major drivers of changes in arthropod populations. This study contributes to identifying the effects of urban sprawl on arthropod diversity and documenting the multiple functions of farm landscapes in peri-urban regions. These cities create strong and ever-increasing pressure on farmland. The urban population of Asia is expanding by more than 45 million residents annually, and this causes a loss of agriculturally productive land of more than 10 km2 d−2. Growing metropolitan regions often penetrate important agricultural areas in emerging peri-urban zones where rural and urban human activities are closely intermingled to form a seemingly chaotic land use pattern3 and create an increase need for food as well as causing poverty and environmental degradation5. Recent studies have shown that the spatial configuration of farmland in peri-urban areas can improve environmental quality6 and help to create an amenity-rich, biodiverse landscape, and to provide outdoor spaces for the urban population8. However, the decline of biodiversity in this type of peri-urban farmland has altered the provisioning of ecological services in agricultural ecosystems; these ecosystems mediate energy and material fluxes or alter abiotic conditions9. But these ecological services suffer in the considerably simplified landscape pattern caused by urban sprawl and the progressive intensification of agriculture10.Asia has become strongly urbanized with 60% of the world’s megacities, those defined as cities having a metropolitan area with more than 10 million inhabitants, located in Asia11 with decreasing non-cropped habitats12. These non-cropped habitats are primarily comprised of trees, hedgerows, and grassy margins and their amount, quality, and spatial configuration can have strong implications for the delivery and sustainability of various ecosystem services15. The presence of a complex landscape with a high proportion of non-cropped habitat is more beneficial than uniform landscapes for maintaining biodiversity, and the focus of attention and research is currently on the effects on biodiversity from the proportion16 and diversification17 of non-cropped habitats. Conditions in non-cropped habitats may provide a wider variety of resources for arthropods than the crop fields18. Retaining viable arthropod populations in farmed landscapes in critical for sustaining the provision of ecosystem services19. Non-cropped habitats in peri-urban farmed mosaics may help to maintain landscape heterogeneity and sustain diverse arthropod assemblages20. Characterizing the dynamic changes of urban sprawl that has affected on landscape patterns of non-cropped habitat in turn has affected arthropods assemblage in peri-urban farmland is important. However, rather few studies have showed the feature of peri-urban farmland arthropod biodiversity in process of urbanization21. In addition, relationship of arthropod biodiversity and the pattern of non-cropped habitat which effected by urban sprawl remain poorly understood22.Related research has shown that farm landscapes surrounding cities tend to become fragmented and discrete units that suffer from homogenization23. Thus, arthropod assemblages may vary in different farmland landscapes that are affected by urban sprawl. It could be hypothesized that arthropod assemblages should be the lowest in most urbanized areas. Based on this hypothesis we analyse: (1) the effects of the intensity of urbanization at multiple scales  on the species richness and abundance of arthropods, (2) the importance of non-cropped habitats to retaining arthropod diversity, and (3) the relationship of farm landscape metrics and arthropod diversity. We expect the findings of this paper to help improve the development of specific approaches to the controlled development of mixed urban-rural landscapes to support the sustainability of urban areas.The main objective of our research was to assess the value of non-cropped habitats that were affected by urban sprawl for sustaining arthropod diversity in peri-urban farmed mosaics. In addition, our study was also aimed at identifying the landscape metrics associated with the spatial heterogeneity of farmland at different distances from a city centre, a factor that influences the structure of arthropod assemblages. The presence or absence of non-cropped habitat in an intensively farmland landscape significantly affects the biodiversity of arthropods2 and has an average elevation of 58 m. The continental monsoon climate in this north temperate zone features four distinct seasons with a mean annual temperature of 7.5 °C and mean annual precipitation of 672.9 mm. The peri-urban regions of Shenyang have continued to extend outward and have been powerfully disturbed by cities and humans with an acceleration of urbanization since the establishment of Shenbei New District in 2006. Shenbei New District is a typical pri-urban area in northeast, China.The study, conducted in peri-urban regions of Shenbei New District  in Shenyang city, Liaoning, China, covered a total area of 10,980 km24  and had little or no influence from urban areas based on their land-use types.For the present study, Shenbei New District was divided into three regions based on landscape disturbance density models24 Fig.  from CheSurveys were performed during the summer  and autumn  of crop growing season in 2013 and 2014, in a typical peri-urban region, selected from urban areas of Shenyang city toward to the administrative boundary of the Shenbei New District. These survey sites were parts of three zones: the inter-urban fringe , outer-urban fringe , and rural areas , within 0–13 km, 13–25 km, and >25 km from urban areas of Shenyang, respectively. The main non-cropped habitat types were classified as either grassland  or woodland  based on vegetation form.24.Arthropods were surveyed in 30 sampling areas (ten areas in each zone) that were selected randomly. Each area had three sampling points, one each in arable land, grassland, and woodland. Three independent 25 × 25 cm quadrants were selected as repetitions for each sampling point26. Arthropods were taxonomically identified at the order level in all cases and at the lowest taxonomic level when possible . Species richness of arthropods was expressed as the mean number of taxa at each sampling point. Arthropod abundance was also measured.As much as possible, potholes, earth mounds, slope, rocksloping areas, rocks, roots, and so on were avoided. The method of Hand picking was adopted to takefor collecting samples of arthropods from three land-cover classes: arable land, grassland, and woodland. The sample specimens were placed into a 75% ethanol collection bottle brought backbottles and returned to the laboratory for identification2), large patch index , edge density , landscape shape index , Shannon diversity index 27 and, aggregate index 28. Three land-cover classes were identified including arable land, grassland, and woodland. Classification was based on the Current Land Use Classification in China (GB/T21010-2007). Land use data (1:10000) for 2012 was obtained from the Shenbei Land Resource Bureau.Landscape heterogeneity was described by calculating the landscape metrics of land-cover types surrounding each field with sampling points. Landscape metrics included patch density  interface with QGIS (www.qgis.org) software. Both landscape- and class-level metrics were computed using land cover grids based on the eight-cell rule for patch neighbours, which considers both orthogonal and diagonal cells as neighbors30.The landscape metrics listed above were computed using a FRAGSTATS31. The significance of observed differences in arthropod richness and abundance between the three urbanization regions analysed in the present study (P < 0.05) was evaluated by visually comparing rarefaction curves and their associated 95% confidence intervals. Estimated arthropod richness and abundance were calculated by using an abundance-based coverage estimator32.Differences in arthropod richness and abundance in the different regions were compared by using a sample-based rarefaction procedure, where individuals are set as samples and curves are then calculated by using the Mao Tau estimator33. Mixed models were used to test for the effects of urbanization within the three zones . Sampling times were not used in the models as co-variables without having significant effects in the arthropod richness and abundance. Sampling points within each region were included as random variables in the mixed-effected models to account for the experiment design. Landscape metrics were included in the model as fixed explanatory variables at each zone. Fixed effect variables were the habitat types within fields, including arable land, grassland and woodland. A simplification model was applied by first removing the non-significant interaction terms . Subsequently, non-significant main effects were removed only when they were not involved in a significant interaction34. Spatial autocorrelation analysis showed no significant values for Moran’s I Coefficient for richness and abundance between plots , because linear mixed effects models were applied to each plot individually. All analyses were performed with R software.The explanatory factors that have contributed to arthropod diversity and abundance were analysed with linear mixed-effects modelsAccording to the Fig. 17.A total of 18 species were identified from the 3102 arthropod specimens captured , II (29% of 14 sp.), and III (43% of 14 sp.), respectively. The accumulation curve of the three regions had similar increasing rate of species numbers found per zone . Regarding the landscape metrics of non-cropped habitats in zones II and III, different landscape patterns maintained similar levels of richness of arthropods. Generally speaking, arthropod richness in zone I was similar to that in zones II and III. Thus, arthropod species richness was negatively associated with urban sprawl in both heterogeneous and homogeneous non-cropped habitats.The species richness of arthropod assemblages has no obvious change in three zones Fig. . MeanwhiThe patterns of arthropod abundance were obviously different to those observed for species richness. Zone III had the greatest abundance of arthropod individuals, while this was significantly lower in zone II and the increased slightly in zone I. In arable land, arthropod abundance was higher in zone III 62.2 ± 8.4) than in zones I (29.9 ± 9) and II (19.9 ± 5.2) Fig. . In gras.2 ± 8.4 35. The present study demonstrated that the isolation of non-cropped habitats contrasted with agricultural biodiversity conservation. In the present study zones, I, II, and III were experiencing different stages of urbanization. That is, the intensity of disturbance varied in different areas based on the nature of ongoing urbanization, which was affected by the city of Shenyang during its northward expansion. Farmland in zone I had already established a new micro-farm landscape pattern under the background of the landscape scale pattern of urbanization. Zone I had experienced strong levels of disturbance from urbanization and this disturbance gradually tended to create a new ecosystem, which gave the non-cropped habitat a certain level of connectivity and complexity. Zone I was in the formation stage for the corresponding relationship between the proportion of non-cropped habitat patches and individual number of arthropods in zone I. Most areas of zone III were still experiencing weak or none urbanization, where the farm landscape was barely affected by urban sprawl. In zone III the original and relatively stable farmland landscape was being maintained, and relatively strong connectivity and complexity remained in the non-cropped habitats of zone III. Therefore, the present study resulted in the same conclusion of another study that showed heterogeneity was associated with non-cropped habitats as a key driver of arthropod population in farmland mosaics18. Zone II was experiencing intense urbanization. Here the non-cropped habitats were unstable because urbanization constantly fragmented and disturbed farmland, which has resulted in disturbance of the landscape pattern of non-cropped habitats. Although zone II has relatively more non-cropped habitat patches in an unstable farm landscape, most were not suitable for arthropod survival, which has been a main cause of decreased arthropod abundance.Developing ecological corridors that are designed to connect residual natural and semi-natural habitats in the farm landscape could help to increase landscape connectivity and to conserve biodiversity in a larger range of farm landscapes37. The disturbance, displacement, and remodelling of landscape structure in non-cropped habitats by urbanization to not all the high level of heterogeneity of farmland to be maintained, which affected the behaviour and movement of arthropods39. In zone I, the process of urbanization in the city had been completed, the remnant of farmland was small and relatively stable as a farm landscape embedded within the urban system, which allowed zone I to retain a relatively high level of patch density and connectivity. Retaining a landscape pattern with non-cropped habitats may help to maintain the heterogeneity of the farm landscape and, therefore may also help to sustain diverse arthropod assemblages. The abundance of arthropods decreased with the decrease in the diversity of non-cropped habitats in zone II because human activities had intensely disturbed the landscape structure of non-cropped habitats, and then reduced the function of non-cropped habitats as refuges and food resources. Furthermore, dispersion and fragmentation of non-cropped habitats resulted in a more intense decrease in arthropod abundance in zone II. In zone III, the farmland mosaics comprised of different non-cropped habitats usually supported relatively high levels of overall arthropod abundance, which resulted from the traditional agriculture structure that had experience no or little influence from urban sprawl. Therefore, in zone III future urban sprawl may disturb the farm landscape and result in a further decrease in arthropod abundance.The landscape pattern of non-cropped habitats is shown in Fig. It is worth noting an obvious change in arthropod abundance had occurred, although there was no obvious change in arthropod species richness in zones I to III. The field investigation revealed that all three zones had the same non-cropped habitat types. The non-cropped habitat type was a major determinant of arthropod species richness in the peri-urban farm landscape because this type of habitat had similar micro-environmental conditions prevailing in all three zones.17. Accordingly, after a disturbance caused by changing stages of urbanization, arthropod species may persist in peri-urban farm landscape through meta-population effects. Nevertheless, we do not know which groups of arthropods species may suffer or benefit from the habitat alteration effected by urbanization. In this regard, future research that identifies species with different traits will be able to provide a finer understanding.In our study, the non-cropped habitat landscape pattern in zone II was more isolated, regular, dispersed, and diverse than similar landscape patterns in zones I and III. Arthropod richness had no obvious change from zones I to III. Arthropod abundance was lowest in zone II, and was significantly lower than in zone III, in rural areas. Therefore, we found that urban-sprawl had negative effects on arthropods, particularly those associated with disturbed non-cropped habitats and weakened farmland heterogeneity. The disturbance dynamics in non-cropped habitats require arthropods to re-establish their populations in these areas annually from surrounding landscape via dispersalHuman activities in peri-urban areas had a clear and significant impact on farmland ecosystems, and the speed of human interference was much larger than the speed of recovery in farm ecosystems with an increase in non-cropped habitats during urbanization. Hence, in order to maintain farm biodiversity and improve the ecological quality of cultivated land it is often necessary to increase the diversity and complexity of farm landscapes. This can be done by simultaneously restricting urban expansion and increasing the proportion of non-cropped habitat areas. To increase the availability of non-cropped habitat, it may be necessary to have clear limits on land use. For example, urbanization may need to be limited in each area to maintain a desired proportion and type of landscape structure in non-cropped habitats.Additionally, a need may exist to monitor and evaluate the ecological quality of farmland and the multi-functional state of habitats that surround cities. This can be done by considering arthropods and other species groups as biodiversity indicator organisms during farmland urbanization, such as monitoring land consolidation, the development of primary cultivated land and so on, in the future.


Bond strength of adhesive layer can absorb unwanted stresses of polymerization shrinkage in composite resin restorations; increased microshear bond strength can prevent failure of restoration materials, the purpose of this study was to evaluate the effect of adhesive layers on microshear bond strength of nanocomposite resin to dentin.Two different types of adhesive systems: universal adhesive (ExciTE) and newly developed adhesive (Nano-Bond), and one type of light-cured resin restorative material (Nanocomposite resin) were used in this study. The occlusal surfaces of extracted human molar teeth were ground perpendicular to the long axis of each tooth to expose a flat dentin surface. The adhesives were applied on dentin surfaces (single application or double application). Nanocomposite resin was then placed and light cured for 40 seconds. After 24 hours of immersion in water at 37°C, then subjected to thermocycling before testing, a microshear bond test was carried out. The data were analyzed by a two-way ANOVA. For comparison between groups, Tukey’s post-hoc test was used.The mean bond strengths of ExciTE and Nano-Bond adhesives with a single application were 8.8 and 16.6 MPa, respectively. The mean bond strengths of ExciTE and Nano-Bond adhesives with double application were 13.2 and 21.8MPa, respectively. There were no statistically significant differences in microshear bond strengths between the single application of Nano-Bond and the double application of ExciTE adhesives.Microshear bond strength increased significantly as the applied adhesive layer was doubled. Key words:Adhesive, microshear, bond, strength, nanocomposite. Stress arising from polymerization shrinkage is one of the most critical problems with light-activated composites . The comFrom restorative dentistry, the use of bonding agents is known to improve the adhesion of composite resins. The bonding agents create a micromechanical interlock between the dentin collagen and resin by forming the hybrid layers .The adhesives could be unfilled or filled ones. The unfilled adhesives have lower mechanical properties, which could lead to gap formation or recurrent caries at the margin of restoration. So the idea of filled adhesives has been introduced,  which inNano-sized filler was introduced to bonding agents by mean of nanotechnology. Nanofillers technology has been known to increase adhesion to both enamel and dentin and improves marginal integrity .Investigators reported that the application of two coats (double-application) of the all-in-one bonding system successfully increased the bond strength to sound dentin . MorpholThe purpose of this study was to evaluate the effect of the adhesive layers on microshear bond strength of nanocomposite resin to dentin. The null hypothesis was that the bond strengths of ExciTE and Nano-Bond adhesives are significantly different between single- and double-application methods.Two different types of available adhesive systems, ExciTE  and Nano-Bond adhesives , and one type of nanofilled composite  were used in this study. ExciTE is a fourth-generation universal adhesive system and Nano-Bond is fourth-generation newly developed adhesive system.Twenty caries-free freshly extracted human molar teeth were collected for use in this study. The teeth were cleaned by ultrasonic scalers and stored in distilled water at 37°C before testing. A dentin slice, approximately 1.0mm thick, was cut perpendicular to the long axis of each tooth from the upper-middle coronal portion region using a low-speed diamond saw  under water coolant. The occlusal surfaces of slices were ground with silicon carbide paper up to #600 grit to expose a flat dentin surface -16.The dentin slices were divided into two main groups (containing 10 each) according to the type of adhesive system. Group A: ExciTE adhesive and Group B: Nano-Bond adhesive system. Each group was further subdivided into two subgroups (containing 5 each) according to layers of adhesive system. Subgroup 1 (A1 & B1): one layer of adhesive systems, and Subgroup 2 (A2 & B2): two layers of adhesive systems.Each dentin slice was acid etched using 37% phosphoric acid gel for 15 seconds. Then the dentin slices were rinsed with water spray and dried with an oil-free stream for five seconds. Primer was applied on the etched dentin surfaces by using the applicator brush. The excess primer was removed with a dry applicator brush, but the surface had a very wet appearance. Then they were light cured for 10 seconds with light emitting diodes (LED). The adhesives were applied on the dentin surfaces by the appropriate manufactures’ instructions or by an experimental method (single or double application). The adhesives were applied to the entire dentin surface and air-thinned for 15 seconds. A gentle stream of dry air was applied to disperse the material into a thin, uniform, shiny surface and, prior to irradiation, three or four cylinders  of Tygon® microbore tubing  were placed on the flat dentin at different locations. The adhesive was then light-cured for 10 seconds with LED. Specimens with thick adhesive layers were produced by the application of one additional coat of adhesive. Each layer was light-cured separately for 10 seconds.After irradiation, each tube was filled with nanofilled composite resin and then light-cured for 40 seconds with the tip as close to the surface as possible. Curing radiometer equipment was used to ensure steady light intensity throughout the polymerization of all specimens. All restorations were finished and polished with a set of solfex discs . The specimens were stored in moist conditions at a room temperature of 23°C for one hour prior to removing the tygon tubing.The specimens were immersed in water at 37°C for 24 hours then subjected to thermocycling to simulate clinical thermal stress conditions before testing, according to the American National Standards Institute/American Dental Association (ANSI/ADA)  and the The resin cylinders were then subjected to the microshear bond test . Each deP<0.05) between group B2 and group A1. Microshear bond strength was increased for the specimens that received two layers.The mean percentage for the tested adhesives (ExciTE and Nano-Bond) with different interactions is presented in Dentin adhesives are currently available as three-step, two-step, and single-step systems, depending on how the three cardinal steps of etching, priming, and bonding-to-tooth substrates are accompanied or simplified. Three-step systems involve etching with an acidic condition, priming with hydrophilic resin in solvent, and bonding with an unfilled or lightly filled resin .The major goals of using dentin bonding systems are to enhance the bonding strength between the resin and the tooth structure, increase the retention of restoration, reduce the microleakage across resin-dentin interfaces, and scatter the occlusal stress .in-vitro mechanical test became of utmost importance to evaluate and compare bond strengths of adhesive systems to enamel and dentin. The most commonly employed test setup for this purpose were tensile and shear tests  between cavity walls and the adjacent composite. This layer could resist the polymerization shrinkage stress of the resin composites and absorb the shock produced by occlusal loads and thermal cycling .According to many investigators ,9 the usAdhesive systems have the acidic monomer ingredient, which demineralizes the subsurface of the dentin, removes or modifies the smear layer, and improves the infiltration of the adhesive resin through residual smear layers into underlaying dentin . These aMultiple applications of adhesives are considered to be effective to prevent the above-mentioned defects of resin bonding and improve the bond performance to dentin; ,12 thereP>0.05). The bond strength of ExciTE and Nano-Bond adhesives were increased with a double application as compared with that of a single application. Nara et al. (In this study, microshear bond strength of ExciTE and Nano-Bond adhesives showed a significant difference between single and double layers (a et al.  reporteda et al. .In this study, Nano-Bond adhesive showed greater microshear bond strength than that of the ExciTE adhesive. This may be contributed to the lightly nanofilled and decreased pH of the Nano-Bond adhesive. Low pH will greatly influence the demoralized degree of tooth substrate. So, the etching of Nano-Bond adhesive is strong enough to completely remove the dentin smear layer. This leads to sufficient infiltration of resin demineralized dentin and increases the bond strength.


The current study aimed to explore age-variant trait differences of cortical gray matter volume (GMV) in a unique sample of first-episode and treatment-naïve patients with schizophrenia. A total of 158 subjects, including 26 adolescent-onset patients and 49 adult-onset patients as well as 83 age- and gender-matched controls were scanned using a 3T MRI scanner. Voxel-based morphometry (VBM) following Diffeomorphic Anatomical Registration Through Exponentiated Lie algebra (DARTEL) was used to explore group differences between patients and controls in regional GMV. We found that patients with schizophrenia had decreased GMV in the left parietal postcentral region that extended to the left frontal regions, the right middle temporal gyrus, the occipital lobe and the right cerebellum posterior pyramis. Further analysis showed a distinct pattern of gray matter alterations in adolescent-onset patients compared with both healthy controls and adult-onset patients. Relative to healthy controls, adolescent-onset patients showed GMV alterations in the left parietal postcentral gyrus, parahippocampal gyrus and right cerebellum posterior pyramis, while GMV deficits in adult-onset patients were focused on the cingulo-fronto-temporal module and right occipital regions. Our study identified differential cortical gray matter deficits between adolescent- and adulthood-onset patients with schizophrenia, which suggests that the cortical abnormalities in schizophrenia are likely adjusted by the developmental community structure of the human brain. However, the results are inconsistent and make it difficult to reconcile the notion that different cerebral regions may be linked with the disorder in different studies. While Matsumoto et al. observed that patients with schizophrenia have reduced gray matter volume (GMV) in temporal lobe regions4, others have reported increased GMV or no structural differences at all in schizophrenic patients6. Possible explanations for the inconsistent findings are the varying of neuroimaging methodologies adopted in studies and the heterogeneity of the disorder itself7. Furthermore, it is not clear if and how these abnormalities are adjusted by the brain’s developmental process.Previous studies have shown cerebral cortical gray matter abnormalities in patients with schizophrenia8. However, the majority of imaging studies in schizophrenia have been confined to adult subjects due to the availability of the sample9. In contrast, there have been few studies that have explored whole-brain changes in early-onset patients with schizophrenia on a voxel-by-voxel basis10. Moreover, the structural cerebral changes revealed in early-onset schizophrenia have previously shown great inconsistencies. For example, Matsumoto et al. reported that the total volume and GMV of the right superior temporal gyrus was significantly lower in patients with schizophrenia than in healthy controls4. Taylor et al., however, found increased volume in the temporal lobe regions of childhood-onset patients with schizophrenia (COS)11.Studying adolescent-onset patients with schizophrenia offers a unique approach to understanding the pathophysiological process of the disorder through exploring the interplaying outcomes of disease-associated pathology and age-related brain development13. Studies have also found that the synaptic density is increased above adult levels during early postnatal brain development13. In addition, the synapses go through a process with brain regions responsible for primary motor and sensory functions maturing earlier than those serving higher-order functions15. Thompson et al. reported that there are parietal and motor cortex deficits at the earlier stage of schizophrenia at baseline, while the deficits of prefrontal, supplementary motor and temporal deficits appeared or increased over the course of the illness16. Alexander-Bloch et al. found that patients with COS showed a converged brain development trajectory with controls in the parietal regions while remaining divergent in the anterior  regions, with a pattern similar to that seen in adults with schizophrenia. This may indicate that patients with adult-onset schizophrenia have undergone a similar process of parietal-frontal wave deficits prior to the onset of illness, while patients with child-onset schizophrenia may have an inflation of the progressive pattern of normal posterior-anterior development17.Compared with the developmental course of white matter, cortical gray matter follows an inverted U-shape trajectory with greater regional variation18. In early-onset schizophrenia, investigations using magnetic resonance imaging (MRI) have suggested that during childhood, the temporolimbic and cingulate brain regions do not show volumetric changes19. Greenstein et al. performed a study with repeated MRI scans in 70 patients with COS aged 7–26 years old and reported a reduction of overall mean cortical thickness, especially in the prefrontal and temporal regions20. However, other researchers have found that early-onset patients with schizophrenia have parietal brain region deficits extending anteriorly into the temporal lobes and reaching the cingulate gyrus16. The difference between adult- and early-onset schizophrenia, in terms of the relationship between progressive changes and clinical correlates, may occur as a result of different brain development stages at the time of the first episode. In addition, a few studies have found that individuals with adult-onset schizophrenia have more widespread white matter deficits in the parietal lobe compared with adolescent-onset patients21.Age-related changes in gray matter throughout normal adolescence might be dynamic, with substantial thinning of cortical gray matter starting initially in primary areas and occurring later in the secondary cortices of the frontal and parietal lobes and finally in the temporal lobes22, with the aim of assessing the effect of age at the onset of schizophrenia on gray matter abnormalities and identifying the gray matter difference between adolescent- and adult-onset patients.It should be noted that most longitudinal studies of first-episode psychosis comprise both adolescents and adults, making it difficult to determine the influence of normal development, and the effects of antipsychotic treatment on the results prior to scanning. Therefore, to avoid these confounding factors and minimize the impact of illness duration and medication on patients, our current study focused on a unique sample of schizophrenic patients who were experiencing their first episode and were treatment-naïve prior to scanning. We adopted an individualized Diffeomorphic Anatomical Registration Through Exponentiated Lie algebra (DARTEL)-based template to provide more precise spatial alignment2 = 0.11, P = 0.75), age  or education years  between patients and controls.The demographic characteristics and clinical information of the participants are shown in Table Compared with their matched controls, there were no significant differences in sex or age in the adolescent- and adult-onset groups, except fewer education years in the adolescent-onset group  and smaller total gray and white matter volumes in the adult-onset group . There were no significant differences in PANSS, GAF and DUP scores between adolescent- and adult-onset patients with schizophrenia . In the adult-onset patients, the GMV of the right posterior cingulate was positively correlated with the paranoid subscale  and the activation subscale . There was also a significant positive correlation between the GMV of the left superior frontal gyrus and depression subscale scores . However, above significant correlations were not maintained after Bonferroni correction for multiple tests.In the present study, by studying a unique sample of first-episode and treatment-naïve patients with schizophrenia, we found that patients with schizophrenia had decreased GMV in the left parietal postcentral region extending to the left frontal regions, right middle temporal gyrus, occipital region and right cerebellum posterior pyramis. Most importantly, with a voxel-based morphometry analysis approach, we demonstrated for the first time a distinct pattern of gray matter alterations in adolescent-onset patients compared with both healthy controls and adult-onset patients. Relative to healthy controls, adolescent-onset patients showed GMV alterations in the left parietal postcentral gyrus, parahippocampal gyrus and right cerebellum posterior pyramis, while GMV deficits in adult-onset patients focused on the cingulo-fronto-temporal module and the right occipital regions.3. Our findings indicated reduced GMV in the parietal, frontal, temporal and occipital cortices in first-episode and treatment-naïve patients with schizophrenia, which is consistent with previous studies, including our own23. These results implicate a wide range of brain changes within the cortices during the earlier stages of the illness and without the effects of antipsychotic medication24.Significant changes in cortical gray matter have been demonstrated in a large number of studies on schizophrenia and confirmed by meta-analysis21. However, because regional alterations of GMV have been frequently observed in various brain regions of early-onset patients rather than being limited to the above brain regions alone26, and because abnormal structural changes of gray matter are deemed to be a fundamental part of the pathophysiology of schizophrenia, the observation of gray matter abnormalities in cortical regions  in individuals with adolescent-onset schizophrenia in the present study may indicate the brain areas in which the disorder originates. However, our studies were not completely consistent with findings from some previous studies on early-onset schizophrenia27, which have shown that frontal gray matter loss may be present in children and adolescents. The difference between studies may be due to the samples included. Our study included adolescent-onset patients only, while others have grouped childhood- and adolescent-onset schizophrenia patients together. Although both childhood-onset and adolescent-onset patients are usually defined as early-onset schizophrenia, it is likely that they have significant differences in precursors, disease course and neurodevelopment.We were particularly motivated by the issue that neurodevelopmental processes continue throughout late adolescence and that an episode of the illness may affect the pattern of GMV alteration in schizophrenic patients, and therefore, we explored the effects of age of onset on GMV in adolescent- and adult-onset subgroups of patients. Using voxel-based analysis, we revealed a regional reduction of GMV in the left parietal postcentral gyrus and parahippocampal gyrus in adolescent-onset patients. This result is consistent with some previous studies in patients with early-onset schizophrenia29. In the present study, we also found reduced GMV in the right cerebellum posterior pyramis in adolescent-onset patients, which is in line with previous findings from earlier studies30. The vermis has a connection with the thalamus and limbic system via the fastigial nucleus31, and abnormality in the cerebellum vermis may disrupt the function of the thalamus-limbic-frontal circuit in patients with schizophrenia32. Our results further confirm the existence of structural abnormality of the cerebellum posterior pyramis in the early stages of the disease.The cerebellum has been suggested to play an important role in the pathophysiology of schizophrenia17, in the current study, we found the adult-onset patients with schizophrenia showed more widespread GMV reductions than did adolescent-onset patients. This reduced GMV was noted in the cingulo-fronto-temporal module and occipital regions in addition to parietal regions in adult-onset patients with schizophrenia. In addition, a study by Kyriakopoulos et al. found that white matter deficits presented predominantly in the frontal and temporal regions in adult-onset patients with schizophrenia and less in the parietal lobe of adolescent-onset patients, suggesting that the abnormal white matter in the frontal regions in schizophrenia may be related to the developmental stage at the time of illness onset21. Our findings indicate a similar pattern to that found by Kyriakopoulos et al. with respect to gray matter abnormalities. Anatomically, it has been shown that the anterior cingulate cortex has strong reciprocal connections with the superior temporal cortex33. This result fits with the proposal that young adult patients with schizophrenia show significant cortical thinning within the occipital, cingulate and frontopolar cortices34, and it is also consistent with evidence that age-constant deficits in cortical thickness overlap significantly with the cingulo-fronto-temporal module in schizophrenia17. Interestingly, a previous study showed that the initial and severe reduction in the cortical thickness of frontal and temporal lobe regions in COS patients may partially be “normalized” in early adulthood21. Furthermore, reduced cortical thickness has been reported in repeated scans from 70 patients aged 7–26 years and diagnosed with COS35. In the normal brain, partial GMV peaks at approximately 11 years old, whereas GMV in the temporal occipital module peaks at approximately age 16.7 years; there is then a progressive reduction in GMV after the peak17. Studies have also implicated the normal maturational process of synaptic/dendritic pruning during the important time of adolescence, which may offer insight into understanding the neurobiological mechanisms underlying “normalized” changes in adolescence and/or more widespread reductions of GMV in adult-onset patients. Thus, it should be strongly suggested to consider the age of onset of the illness as a significant factor when examining progressive brain changes in schizophrenia.Consistent with previous studies36; however, most previous studies recruited patients with schizophrenia who were being treated with antipsychotics at the time of the scan and therefore could not completely eliminate the effects of antipsychotic treatment39. Second, compared with the classic ROI method, previous studies employing ROIs with pre-specification have done so on a relatively small number of hypothesized brain regions in patients with schizophrenia40. In fact, voxel-based morphometry has proven to be somewhat more sensitive than ROI methods in terms of attempting to systematically review a large body of neuroanatomic literature34.The present study has several advantages. First, we recruited a group of first-episode, treatment-naïve patients with schizophrenia in our analysis to minimize confounding factors due to medication use and the chronicity of the illness. Previous studies have indicated that the cortical volume abnormalities in patients with schizophrenia might have genetic or shared environmental origins rather than being a consequence of drug treatments41. Furthermore, a longitudinal rather than cross-sectional design is required to verify the dynamic changes of GMV in adolescent- and adult-onset patients.The results of this study should be interpreted in the context of the following limitations. Schizophrenic patients and control subjects showed significant differences in terms of educational years. However, we found there was no association between educational years and abnormal GMV, so this difference may not alter the overall significance of our resultsIn conclusion, our research found that reduced GMV in adolescent-onset patients with schizophrenia mainly focused on the parietal cortical regions, while adult-onset patients showed reduced GMV in the cingulo-fronto-temporal module and right occipital regions. The differential cortical gray matter deficits in adolescent- and adulthood-onset patients with schizophrenia may suggest that the cortical abnormalities in schizophrenia are likely adjusted by the developmental community structure of the human brain.42. All of the patients underwent further evaluation of their clinical symptoms using the Positive and Negative Syndrome Scale (PANSS), which provided a total score and syndrome scores for the following categories: Negative, Positive, General, Thought disturbance, Activation, Paranoid, Impulsive aggression, Anergia, and Depression43. All of the items were rated from 1 (absent) to 7 (extreme) according to standardized instructions.A total of 158 subjects participated in this study, including 75 patients with first-episode schizophrenia and 83 healthy controls. All of the patients were recruited from inpatient and outpatient psychiatric units at the Mental Health Centre of the West China Hospital from 2008–2013. The patients were assessed by trained psychiatrists using the Structured Clinical Interview for DSM-IV Patient version (SCID-P) after their first presentation to mental health services. Diagnoses were assigned according to the diagnostic criteria for schizophrenia as specified in the DSM-IV. To be included, an individual was required to fulfill the following inclusion criteria for the present study: age between 14 and 45 years, Han Chinese, right-handed, intelligence quotient ≥70, experiencing their first episode of schizophrenia, and treatment-naïve or no more than 3 days of antipsychotic treatment before clinical assessment and MRI scan. Information on previous treatment, the duration of untreated psychosis (DUP) and the Global Assessment of Functioning (GAF) score were collected at diagnosis44.Healthy participants were recruited between 2008 and 2013 via word-of-mouth or posting advertisements. The inclusion criteria for controls were as follows: age between 16 and 45 years, Han Chinese, right-handed, and intelligence quotient ≥70. All of the controls were screened for a lifetime absence of Axis I illness or DSM-IV psychiatric illnesses with the SCID Non-Patient version (SCID-NP). In addition, healthy participants were interviewed to ascertain that there was no psychiatric illness in their first-degree relatives. Participants with a history of any major psychiatric disorders (such as affective and schizoaffective disorders), intellectual disability, head trauma, any substance abuse (except tobacco), alcohol abuse disorder, any major nervous system diseases, and severe physical diseases were excluded. All subjects were Han Chinese and right-handed as assessed by the Annett Handedness Scale21. Two subgroups of healthy controls were age- and sex-matched with the adolescent-onset and adult-onset patients. Written informed consent was obtained from all participants or their guardians if they were under 18 years of age. All methods were performed in accordance with the relevant guidelines and regulations approved by the Institutional Review Board of West China Hospital of Sichuan University.Patients were assigned into 2 subgroups: patients with adolescent-onset (aged 14 to 17 years) or with adult-onset (aged 18 to 45 years) schizophrenia47. Foam padding and earplugs were used to minimize head movement and scanner noise. High-resolution T1 images were obtained by the 3D spoiled gradient echo sequence (SPGR) from all participants. The protocols used were as follows: TR = 8.5 ms, TE = 3.93 ms, flip angle = 12°, thickness of slice = 1 mm, single shot, field of view (FOV) = 24 × 24 cm2, matrix = 256 × 256, size of voxel = 0.47 × 0.47 ×1 mm3. In total, 156 slices of axial images were collected from each brain. The raw MRI data were checked by two experienced neuroradiologists. No artifacts or gross anatomical abnormalities were observed for any subject48.All scanning was performed on a 3T MRI system  using an eight-channel phased-array head coil without upgrading of the scanner software or hardware except daily management and maintenance during 2008 and 2013 in order to keep data consistence and high qualityhttp://www.cabiatl.com/mricro/mricron/dcm2nii.html). The 3D T1-weighted images were processed using voxel-based morphometry in Statistical Parametric 8 (SPM8) software (http://www.fil.ion.ucl.ac.uk/spm) and ran on the MATLAB 7.3 platform . Next, using the Medical Image NetCDF (MINC) software package (http://wiki.bic.mni.mcgill.ca/index.php/MINC), we adopted the non-parametric non-uniformity intensity normalization technique (N3) to strengthen the non-uniformity of the high-magnetic field signals obtained in the transformed images. Because we wanted to study voxel-wise changes between schizophrenic patients and control subjects across the whole brain, the use of non-linear deformations to register native scans into a common space played an important role in carrying out the process with greater accuracy. Thus, voxel-based analyses using different practical methodologies for the automated segmentation and registration of the brains through a new segment tool implanted in SPM were carried out for the investigation of gray matter morphometry. The DARTEL algorithm, which is an automated, unbiased, and nonlinear template building program, was adopted using a similar strategy to that described by Asami et al.7. The specific procedure was performed as follows: first, gray matter was automatically segmented using tissue-signal intensity values or tissue priors for the distribution of brain tissue type ; second, the gray matter tissue class images were spatially normalized onto the subject-specific template space, and the signal intensity of the normalized maps was modulated using the determinant of the Jacobian of the transformation; third, the population gray matter template was created by the DARTEL algorithm using all of the subject-specific templates of the gray matter tissue images; fourth, the normalized gray matter tissue maps for each subject were spatially and nonlinearly normalized to the population template and then modulated; fifth, the population GM template was aligned to the stereotaxic space using a 12-parameter affine transformation, and all of the individual tissue class images that were residing in the population template space were then co-registered to the stereotaxic atlas space using the same affine transformation; and finally, the gray matter tissue class images were smoothed with a 4-mm FWHM Gaussian kernel. A relatively small Gaussian kernel size of 4 mm was chosen to maintain the sensitivity to detect small brain structural changes in patients and, more importantly, to improve the signal-to-noise ratio. The volumes of gray matter and white matter were estimated from the smoothed images using custom MATLAB code 49.Image files in DICOM format were transformed to NIfTI format using dcm2nii software , was applied to ensure that only voxels within the gray matter mask were analyzed. We set the significant differences at the threshold of P < 0.05 at a FWE corrected cluster level and voxel-wise t > 3.1250. Each individual cluster that showed significant differences between groups was defined as a region of interest (ROI). The GMVs of individual ROIs were extracted from each subject and compared between groups using ANOVAs in SPSS 16.0. To assess the functional significance of GMV alterations in patients, the values of GMV in each ROI were correlated with the PANSS and the DUP scores. The correlation analysis was carried out using Pearson’s correlation or Spearman’s correlation if the variables showed non-normal distribution in SPSS, in all patients (including the adolescent and adult patients) and in the adolescent and adult groups respectively. The significant correlation was set at P < 0.05 (two-tailed) after Bonferroni multiple comparisons.The smoothed images, which were generated from image preprocessing, were entered into two-step analysis. Voxel-wise comparisons of GMV were performed using two-sample t-tests between patients and controls. Because age, sex, and total brain volume are potential confounding factors affecting regional cerebral volumes, their effects were removed by controlling the covariance. The above analyses were then performed in the two onset age-groups (age- and sex-matched) to examine the different patterns between adolescent- and adult-onset patients. Explicit masking, which was constructed from the overall 158 subjects using the “SPM Masking Toolbox” (


Although early presentation to HIV-care is essential to ensure timely initiation of antiretroviral therapy, recent studies have shown that especially migrants present to HIV-care at a later stage of HIV-infection. Currently, thirty percent of all newly diagnosed HIV cases in Germany originate from abroad. So far it is unknown, which specific migrant groups in Germany are particularly at risk for late presentation to HIV-care.We used data from the Clinical Surveillance of HIV Disease (ClinSurv) cohort, a multi-centre observational cohort (01/01/1999 and 31/07/2013) and included treatment-naïve patients with valid information on country of origin and date of enrolment. Migrants were patients with country of origin outside Germany. We compared time trends for percentage of AIDS (CDC Stage C) and mean CD4-count at enrolment between migrants from Western Europe (WE), Central Europe (CE), Eastern Europe (EE), Sub-Saharan Africa (SSA), South East Asia (SEA) and non-migrants using multivariable regressions. Male non-migrants with mean age of 38-years constituted the reference group.SSA: 1.44, 95%-confidence interval (95%-CI):1.12–1.84) and SEA-migrants . In addition, female SEA-migrants, were more likely to present with AIDS than their male counterparts . Mean CD4-count at enrolment was lower for SSA- (Mean CD4-count ratio (IRR):0.72; 95%-CI:0.64-0.82) and SEA-migrants . Over time, it increased in non-migrants and CE-migrants , whereas no increase was seen for SEA and SSA.In total, 10,211 patients fulfilled the inclusion criteria, of which 2784 were migrants . The percentage of patients with AIDS at enrolment was higher in SSA (Odds Ratio (OR)SSA and SEA-migrants in Germany present to HIV-care at a later stage of HIV infection than non-migrants. Additionally, previous research found a higher risk for late HIV-testing for migrants. Collecting information about the arrival date of migrants in Germany in the HIV notification system would help to understand to which extent these problems could be tackled in Germany. Moreover, participatory approaches for HIV-testing and care as well as research regarding knowledge, behaviour and attitudes towards these topics for SSA and SEA migrants should be expanded. According to the latest International Migration Report of the United Nations, there are over 200 million migrants worldwide, of which 10 million reside in Germany [In Europe between 2007 and 2012, 40% of the cases of Human Immunodeficiency Virus (HIV) reported to the European Centre for Disease Prevention and Control (ECDC), originated from a country outside the country where their HIV infection was diagnosed .Generally, HIV-testing in Germany is offered free-of-charge. In addition, anonymous testing is possible at most local public health voluntary counselling and testing sites and at several non-governmental organizations. HIV-testing for pregnant women and patients with suspected HIV-infection at the general practitioners and specialists is covered by statutory health insurances .German-Austrian antiretroviral treatment (ART) guidelines for HIV-infection for adults recommended that ART should be initiated in all symptomatic patients especially after the onset of at least one illness defining acquired immunodeficiency syndrome (AIDS) , 6. FurtART in Germany is offered to all HIV-positive patients. It is fully paid by statutory health insurance, which postulates treatment by specialized physicians . In ordePast studies on clinical features in migrants in Germany have already shown an association of migration status with late presentation for HIV-care . Howeverst January 1999 and is a network of 15 HIV treatment centres and university clinics in major German cities, which collect data on about 19.000 HIV-positive patients (as of 31 July 2013) with unknown date of HIV-infection. For each patient who seeks care in a participating centre, information on age, sex, country of origin (collected by the treating physician according to patient’s information) and transmission group category was reported at enrolment in ClinSurv. Clinical parameters  stage, immunological and virological status  of HIV infection as well as information on ART  are recorded upon enrolment and updated for every date in which the patient visits the centre after enrolment. The collected data is sent biannually in an encrypted format to the Robert Koch Institute, where it undergoes verification and quality checks [We used data from the Clinical Surveillance of HIV Disease (ClinSurv) cohort. This open multi-centre long term observational cohort was established on 1The ClinSurv cohort collects anonymized data on all HIV-patients, who are in medical care in these 15 participating centres.Authors from the Robert Koch Institute, as the data processing site, only had access to anonymized data and had no access to identifying information. All data analyses were performed on the anonymized data set. There were no permissions required to conduct scientific analyses.For our analysis we extracted all entries of patients enrolled in ClinSurv between 1 January 1999 and 31 July 2013, who were older than 15 years and treatment naïve at enrolment in ClinSurv and where the definite time of enrolment into ClinSurv was known. Furthermore, only patients, where sex and country of origin were available in the database were included. We excluded patients who acquired HIV-infection through haemophilia, mother-to-child-transmission or by occupational exposure (rare transmission routes).As described above, a migrant was defined as a patient with a reported country of origin outside Germany. Migrants were further categorized in migrant groups by regions of origin according to the World Health Organization (WHO) region definitions .AIDS was defined by CDC Stage C diagnosis. Here the date of diagnosis before enrolment or within 45 days after enrolment was classified as presenting with AIDS at enrolment into the cohort. We obtained the CD4-count in cells/μl of a patient when presenting to specialized care by using the CD4-count measured at the enrolment of the patient into ClinSurv.In order to get an overview of the study population, we analysed the most frequent regions of origin for migrants descriptively. For non-migrants and migrants by region of origin, the median age, percentage of males, most frequent risk groups and mean CD4-counts and proportion of patients with AIDS were compared. We performed rank sum and Chi-squared tests to test for differences in sex and age at ART-initiation between migrants overall and non-migrants.We performed multivariable regressions to estimate the difference in CD4-count and percentage of patients with AIDS prior to enrolment into ClinSurv between non-migrants and the five most frequent migrant groups. The other migrant regions, which were represented by less than 200 enrolled patients over the timespan 1999 to 2013, were excluded from the regression models. Instead of the continuous age variable used in the descriptive analysis, for the regression analysis, age groups  were formed in order to consider also non-linear effects of age. Regarding CD4-count we performed negative binomial regressions including only patients with available information on CD4-count at their enrolment visit. The obtained incidence rate ratios (IRR) in our model refer to the mean CD4 count ratio. IRR were calculated with their 95%-confidence intervals (95%-CI). In the negative binomial regression, the variance can be estimated through the dispersion parameter. To compare the percentage of patients with AIDS, odds ratios (OR) and their 95%-CI were analysed in logistic regressions. In addition a time-variable was included in the regression models, to describe the trends of these values over the time period 01/01/1999 to 31/07/2013. Male non-migrants in the age group 35–44 years (which included the median age of this subgroup) constituted the reference group. An alpha of 0.05 was considered the level indicating statistical significance.All analyses were performed with the software STATA 14.0.Of 19,256 patients enrolled in the ClinSurv cohort as of 31/07/2013, 10,211 patients fulfilled the inclusion criteria  and were less often male  . While Western and Central Europeans show a more similar distribution of these characteristics to non-migrants, migrants from Eastern Europe display the lowest age at enrolment. Migrants from Sub-Saharan Africa are least frequently male (39%).Migrants overall enrolled in the cohort at a significantly younger age (median age: 34 years) than non-migrants (median age: 38 years) -transmission route . For migrants from Eastern Europe, Injecting drug use (IDU) as a transmission route was noticeably higher (27%) compared to other migrant regions . Overall no difference in AIDS at enrolment between male and female patients was found, except for South East Asian migrants, where females are more likely to present with AIDS to specialized care than their male counterparts    for non-migrants and 3%/year (95%-CI: 1%–5%) for migrants from Central Europe. For all other migrant groups, no significant trend is visible  compared to native populations at initiation of ART , 16. ParTo get an insight in the population of people living with HIV in Germany at point in time previous to linkage to HIV-care, namely the first HIV-test in Germany, Zoufaly et al. performed additional analysis of German HIV surveillance data in addition to the ClinSurv analysis . It provResults of participatory research at the Robert Koch Institute among migrants from Sub-Saharan Africa in Germany (MiSSA) show that migrants of this group were often not aware of the anonymous and free HIV-testing offer or assumed that a positive HIV-test will impact their legal status in the country . These tIn contrast to other migrant groups, Eastern European migrants present to care at an earlier stage of HIV-infection than non-migrants in terms of AIDS and mean CD4-count. A reason for this could be that a large part of the HIV-infections in the Eastern European migrant population in Germany is acquired via IDU and MSM transmission. For those risk groups HIV-testing is especially promoted in Germany. Other migrant groups, such as South-East Asian migrants and Sub-Sahara African migrants consist of females, predominantly infected by heterosexual transmission – risk groups, who are not especially targeted for HIV-testing. This fact underlines once more the importance of accounting for the different composition of migrant groups in terms of HIV.According to a recent review on HIV-testing policies in Europe, the German HIV-testing framework, although acknowledging migrants as a vulnerable group for HIV has so far neither issued a tailored recommendation for migrants for HIV-testing within the HIV-testing strategy  nor haveRKI conducted first efforts to establish guidelines for a more tailored HIV-testing framework for migrants in Germany in the MiTest-Study between 2014 and 2015 . This miThe legal and political framework for migrants in Germany, the complex composition of the migrant pool in Germany, the lack of more need-adapted testing offers and culture-related factors (such as lack of HIV knowledge and feared stigmatization) nevertheless still form barriers for migrants’ HIV-test seeking uptake, according to the study.However, when interpreting our results some limitations must be considered.The majority of migrants recorded in the ClinSurv cohort are covered by statutory health insurance and have regular access to the health care system in Germany. As a consequence, the study population lacks information regarding the proportion of migrant people with AIDS symptoms without any access to the German health care system (for example undocumented migrants). The proportion of AIDS among this group might be higher than observed in our study collective.Secondly, small sample sizes of certain migrant groups for example the number of enrolled Eastern European migrants in the cohort, make interpretation of the results for these particular subgroup only possible with caution.Last, no information is available about the arrival date of the migrants in Germany and also no documented information about the first HIV test of the patients. Therefore no conclusions about HIV disease progression since arrival in Germany can be drawn out of the cohort data. As past evidence and current recommendations illustrate, it would be helpful to include information about migrants’ arrival date in Germany into HIV-notification system to close this interpretation gaps in the future , 32).In conclusion, we show that particularily migrants from Sub-Saharan Africa and South-East Asia in Germany enter HIV-care at a later stage of HIV infection than non-migrants. This may be a result of late presentation for HIV-testing, as previous research indicates. Collecting information about the arrival date of migrants in Germany in the HIV notification system would help to understand to which extent this problem could be tackled in Germany.In order to reduce the proportion of late presentation among those migrants groups, the existing participatory approaches for HIV-testing and care should be further strengthened. It is important to disentangle even more the heterogeneous group of migrants, to identify which particular groups still remain unreached by those approaches and identify their specific community organizations in Germany. This opens the door for implementation of knowledge attitude, behaviours and practice studies - similar to the research performed within migrant groups from Sub-Saharan Africa in Germany (MiSSA) - for further migrant groups aiming to develop new tailored strategies, which will improve these groups’ access to HIV-testing and care.


An oxaliplatin-based platinum(iv) drug which specifically binds to albumin after i.v. application led to several complete responses in tumor-bearing mice. iv) prodrugs is a very promising approach to enhance the low selectivity of platinum(ii) drugs towards cancerous tissue in order to reduce the impact on healthy tissue and, consequently, the often severe side-effects. Herein, we report a set of mono-functionalized cis- and oxaliplatin-based platinum(iv) complexes bearing a maleimide moiety, which allows selective binding to serum albumin in the bloodstream. This leads not only to a prolonged plasma half-life by avoidance of fast renal clearance, but also to preferential accumulation of the drug in the tumor tissue due to the EPR-effect. Additionally, analogous succinimide-functionalized derivatives were prepared to verify the influence of the maleimide moiety. First experiments showed that all the maleimide compounds are stable and also possess good albumin-binding properties in whole serum. Further analytical studies on in vivo samples proved the highly increased plasma half-life, as well as tumor accumulation of the maleimide-functionalized substances. In vivo antitumor experiments with CT-26-bearing mice showed that, in contrast to the cisplatin derivatives, the oxaliplatin-based complexes had exceptionally better activity than the free drug resulting in the cure of the majority of treated mice. Subsequent analysis suggested that a distinctly faster reduction as well as reduced tumor accumulation of the cisplatin derivative might explain the worse performance compared to the oxaliplatin(iv) complexes. Taken together, a novel lead platinum(iv) complex with outstanding antitumor activity is presented, which will now be further developed towards clinical phase I trials.The design of targeted platinum( However, the treatment is frequently accompanied by severe side-effects and acquired or intrinsic resistance.3 Aside of cisplatin, only two additional platinum(ii) complexes have been approved worldwide, namely carboplatin and oxaliplatin. While carboplatin is similar to cisplatin in its area of application, oxaliplatin is especially active against colon carcinoma.4 Nevertheless, the low selectivity of platinum(ii) drugs towards cancer cells and cancerous tissue, together with severe adverse effects are still major disadvantages.6 An approach to enhance the selectivity are platinum(iv) prodrugs, which are chemically more inert and thus less cytotoxic. After entering the hypoxic, reductive conditions of the cancerous tissue, they are reduced and release their active, highly cytotoxic Pt(ii)-core (activation by reduction).7 Consequently, the adverse effects on healthy tissue should be reduced. From a chemical point of view, the two additional ligands of octahedral platinum(iv) compounds offer a broad range of possibilities to modify such compounds and to fine-tune their pharmacological properties like lipophilicity or solubility.8 Nevertheless, only a few of a countless number of platinum(iv) compounds prepared ended in clinical trials, with satraplatin as the most prominent representative. Satraplatin reached a clinical phase III study in combination with prednisone, called SPARC (satraplatin and prednisone against hormone-refractory prostate cancer) trial. However, this platinum(iv) drug failed to show convincing benefits compared to standard therapy and therefore satraplatin was not approved for cancer therapy.9 Notably, some studies showed that satraplatin is already unselectively reduced in blood and/or with liver enzymes to the platinum(ii) complex.11Since the discovery of the anticancer activity of cisplatin by Rosenberg iv) compounds. An option would be to exploit tumor-specific properties (e.g. overexpression of diverse receptors) e.g. by using targeting molecules like vitamins or peptides, a strategy known as “active targeting”. However, as such drugs are usually below a size of 60 kDa, they are still susceptive to renal clearance.12 On the other hand, tumor-specific activity can also be achieved by “passive targeting” strategies via the so-called enhanced permeability and retention (EPR) effect.13 This effect can be described by the combination of fenestrated blood vessels together with a lack of a lymph drainage system in the solid tumor tissue, which results in tumor-specific accumulation of macromolecules like nanoparticles and large proteins.16 One of the most elegant and promising ways to exploit the EPR effect is the use of human serum albumin (HSA) as a carrier.18 Abraxane®,20 an albumin-bound nanoparticle formulation of paclitaxel, which was approved for metastatic breast cancer, non-small cell lung cancer (NSCLC) and pancreas adenocarcinoma, as well as the doxorubicin-based drug aldoxorubicin,21 which is currently in clinical phase III studies, are two successful examples. Aldoxorubicin possesses a maleimide moiety, which is known to rapidly bind to the free thiol group (cysteine at position 34)22 of albumin exploiting it as a carrier without the use of exogenous compounds usually used for nanoformulations. With regard to platinum compounds, there are a few publications of platinum(ii) complexes, which possess a maleimide moiety attached to the leaving group.24 The drawback of coupling to a platinum(ii) compound is that in this oxidation state the compounds are quite reactive, especially considering the long plasma half-life of albumin. In addition, a platinum(iv) complex with a highly hydrophobic C16 alkyl chain with non-covalent albumin binding properties has been reported, however, with the disadvantage of low aqueous solubility.25 We recently presented a proof-of-principle for bis-maleimide-functionalized platinum(iv) complexes.26 However, albumin-binding studies indicated that despite the presence of two maleimide moieties, only one albumin molecule is able to bind these prodrugs.26 Thus, the second maleimide is free to  react with other proteins or thiol-containing molecules, which might lead to an uncontrolled pharmacological behavior in vivo. Therefore, we herein report on the first mono-maleimide platinum(iv) complexes not only of oxaliplatin but also of cisplatin. Unexpectedly, only the oxaliplatin derivatives exerted distinctly enhanced activity as compared to the free drug, while the cisplatin prodrugs failed to result in improved anticancer effects in vivo. Moreover, we discovered that small structural modifications on one axial ligand resulted in distinct differences in the anticancer activity of the novel compounds. This finally led to the identification of a highly in vivo-active oxaliplatin-releasing derivative, which is now a lead candidate for further preclinical evaluations.In general, there are several possibilities to improve the selectivity of platinum(ii) precursors for the syntheses of the novel platinum(iv) prodrugs were cisplatin (1) or oxaliplatin (2). In the first step, the platinum(ii) compounds were oxidized with hydrogen peroxide (50%) using either methanol27 or acetic acid28 as the solvent, to yield the unsymmetrically oxidized platinum(iv) precursors 3–6. The maleimide- and succinimide-functionalized ligands were prepared by conversion of the propionic acids to the corresponding isocyanates.26 Finally, all eight mono-functionalized complexes were prepared via reaction of the isocyanate-containing ligands and the hydroxide function of the platinum(iv) precursors 2CH2CH2NH2)Cl2(OH)(OMe)30 and the Pt–O distance of the acetato ligand with ∼2.01 Å is similar to Pt(NH3)2Cl2(OAc)2  was investigated. Therefore, compounds onitored .7–8 reacted faster with HSA than the oxaliplatin-derivatives 11–12. Concerning the methoxido- and acetato-functionalized compounds, no distinct trend regarding albumin binding could be observed. An increase in HSA equivalents also accelerated the binding as expected. In comparison to the previously published data on bis-functionalized maleimide compounds,26 the half-life time of the mono-functional complexes is approximately doubled. This is in line with the lower number of maleimide groups able to bind to albumin.In general, the cisplatin-based compounds 7–8 and 11–12 were bound to the albumin fraction at 7.6 min molecules were measured with size exclusion chromatography inductively coupled plasma mass spectrometry (SEC-ICP-MS) over 21 h. Already at the first scan (∼5–10 min after incubation) ∼50% of the maleimide-containing compounds cysteine . Thus, ting 21 h , proving8, 11 and 12 in comparison to cis- and oxaliplatin was tested against subcutaneous CT-26 tumors in male BALB/c mice . Unexpectedly, despite their similar behavior in the binding studies, distinct differences with regard to onset and strength of anticancer activity between the different maleimide compounds were observed: as such, only the two oxaliplatin derivatives proved to be superior to their respective platinum(ii) compound . This resulted not only in prolonged overall survival but also cured one of the treated animals  in complete remission of one animal, allowing the histological analysis of 3 animals only. In addition, as depicted in 12 were clearly smaller than the ones of the other treatment groups. Microscopy analyses of the tissue morphology (by counting) revealed that all drugs of our test panel induced a significant increase of the apoptotic cell fraction , indicating that the peak of anticancer activity had been reached earlier and the activity was already declining at the time point of tumor collection. Furthermore, the histological evaluation of tumor material was only possible for 3 animals because the fourth mouse already experienced a complete remission at time of section . Noteworthy, in the three remaining tumors treated with 12 a visible reduction in the mitotic cell fraction was still found  and paraffin-embedded tumors were evaluated by H/E stain. In the case of y 12 see : thus, ht day 12 , had lowd Fig. S5. However36 Especially, the enhanced accumulation due to the EPR effect makes this form of tumor-targeting very interesting for the development of novel cancer therapeutics.13 As our novel compounds have a double prodrug nature , we were interested in the drug tissue distribution after treatment. To this end, male CT-26-bearing BALB/c mice were treated once with our compounds followed by sample collection 24 h later. 40 for both platinum(ii) drugs the intratumoral drug levels were distinctly lower than in the liver and kidney . Interestingly, although more than 2-fold higher platinum levels were applied in the case of oxaliplatin and its derivatives (11 and 12) compared to cisplatin and its prodrug 8, the tested organ levels were in a similar range. This indicates that there are per se strong differences in the pharmacological behavior of the oxali- and cisplatin. With regard to the impact of the maleimide moiety, all three platinum(iv) prodrugs showed distinctly enhanced tumor levels in comparison to the respective platinum(ii) compound. However, only in the case of 12, tumor levels significantly exceeded the ones of the kidney and liver (p > 0.05 by one-way-ANOVA), while they were not significantly different or even lower in the case of 11 and 8, respectively. Noteworthy, despite the lower applied dose (equimolar to 3 mg kg–1 cisplatin) in the case of the cisplatin derivative 8, the animals in this treatment group were characterized by organ platinum levels in the range of the oxaliplatin derivatives and even higher platinum content in liver and kidney tissue. This is remarkable as parallel SEC-ICP-MS and flow-injection-(FI)-ICP-MS investigations of the serum samples 24 h after treatment revealed that, in accordance to the applied doses, the total Pt levels of the oxaliplatin derivatives 11 and 12 were more than 2-fold higher than the one of the cisplatin derivative 8 (ii) drugs were already very low.It is well accepted that albumin targeting distinctly impacts on the plasma half-life time as well as tissue distribution.vative 8 . Notably12 and, by this, results in a prolonged plasma half-life time. However, our data indicate that 12 is superior to 11 and especially 8 with respect to tumor-targeting indicating distinct differences in the pharmacological behavior and susceptibility to the EPR effect.In general, the higher tissue and serum levels of the maleimide compounds were not unexpected, as albumin binding of drugs  is known9–10 and 13–14 were evaluated in these experiments (9–10) are reduced much faster than the oxaliplatin-based complexes (13–14), which can be explained by the faster electron transfer via the chloride ligands.41 Additionally, platinum(iv) compounds bearing an acetato ligand  were more stable than those with a methoxido ligand . Comparison of the HPLC data and the NMR measurements showed very similar tendencies. Overall, there is a strong correlation between the reduction data and the in vivo activity. As such, the fast reduction of the cisplatin derivatives correlates with the low in vivo activity of 8 and relatively low plasma levels after 24 h. In contrast, the oxaliplatin derivatives were characterized by very high inertness towards reduction (even more pronounced for the acetato derivative 12) together with higher plasma levels after 24 h and strongly enhanced anticancer activity.As one explanation of the observed differences between the compounds might be altered reducibility, we investigated the reduction rate in the presence of ascorbic acid in phosphate-buffered solution at pH = 7.4 using NMR spectroscopy and HPLC measurements. To reduce the number of additional signals from hydrolyzed maleimide species in the spectra, only the succinimide-functionalized compounds eriments  and S6†.8 was incubated with 5 eq. of HSA to achieve maximal binding and remaining low-molecular-weight platinum was removed by filtration through Sephadex. Subsequently, the obtained HSA-platinum fraction was incubated with 10 eq. of ascorbic acid for 24 h for reduction. After removal of the high-molecular-weight fraction, the solution was analyzed by FI-ICP-MS and RP-HPLC coupled to an ICP-MS instrument. The data clearly revealed the formation of a platinum(ii) reduction product, which elutes at the same time as the cisplatin reference compound  species was only ∼10% of the total platinum content, which possibly can be explained by non-covalent binding of the released cisplatin to albumin.43To further investigate the reduction process, the release of the HSA-bound platinum was investigated. To this end, the cisplatin-based maleimide compound 44 In order to address these issues, more kinetically inert platinum(iv) drugs were developed with satraplatin as the furthest clinically developed representative.45 However, even this promising compound, while being less toxic, failed in a clinical phase III trial, because it did not reach the endpoint of improved overall survival.46 This might also be associated with the fact that despite being a prodrug, satraplatin has no tumor-targeting properties. In addition, it is still questionable, whether the proposed activation of the drug by reduction to the active platinum(ii) complex is a solely tumor-specific process, as there are several indications for reduction of satraplatin e.g. in the red blood cell compartments by reaction with hemoglobin10 or in the liver by metabolizing enzymes.11 Consequently, in the last few years we focused on the development of novel tumor-targeted platinum(iv) drugs.26 In this study, we present the first mono-functionalized maleimide-containing platinum(iv) complexes. The panel of newly synthesized complexes showed that there are distinct differences, e.g. in the in vivo anticancer activity, not only between cis- and oxaliplatin derivatives, but also between closely related compounds, which only differ in one axial ligand. Subsequent analyses revealed that this is most likely not based on the aqueous stability or albumin binding velocity of the compounds. However, strong differences in the reducibility of the platinum(iv) cores were observed, which could provide at least some explanation for the observed effects in vivo. Thus, the cisplatin derivatives were characterized by much faster reduction compared to the oxaliplatin analogues. This is in line with recent literature reports, which show that complexes containing equatorial chlorido ligands have distinctly shorter half-lives in the presence of ascorbate in aqueous solution compared to their oxalato or cyclobutane dicarboxylic acid analogues.48 Noteworthy, the exchange of the methoxido to an acetato ligand further increased the inertness towards reduction. In addition to the reducibility, also the tumor-targeting potential of the albumin-bound drugs 24 h after i.v. administration distinctly varied. This is unexpected as the detected serum platinum levels were in line with the applied drug dose and SEC-ICP-MS serum incubation experiments revealed that the stability of the albumin adducts did not differ between cis- and oxaliplatin derivatives. This indicates that the observed differences in tumor-targeting are not based on interaction with reductants in the serum but on other factors. Hence, for example, the different reducibility of the drugs might impact on their metabolization in the body, which would also fit to the comparably high platinum content of 8 observed in the main organs responsible for drug elimination, namely liver and kidney.Besides the rapid development of drug resistance, adverse effects are still the major limitation for successful chemotherapy in the advanced stage. In the case of platinum drugs especially nephrotoxicity, neurotoxicity, ototoxicity, and myelosuppression are most problematic.in vivo anticancer activity between the two oxaliplatin derivatives 11 and 12 are remarkable. Although they behaved widely similar with respect to their chemical properties under cell-free conditions , compound 12 resulted in the complete and long lasting response of ∼25% of male and ∼75% female animals . In contrast, 11 just led to disease stabilization but not to tumor regression. This is also surprising as both derivatives were present at similar serum levels after 24 h. However, some differences were observed with respect to the tissue levels of the drugs, where 12 was superior to 11. This might indicate that the drugs differ in their extravasation potency e.g. by different EPR efficacy of the drug–albumin-adducts or by different affinity for glycoprotein 18 and 30 (gp18 and gp30), two membrane-associated proteins, which are involved in the recognition of conformationally modified albumin molecules.18 As both fields – albumin metabolism and in vivo activation by reduction of platinum(iv) drugs – are surprisingly unexplored, more in-depth studies are necessary to fully understand the impact of albumin-targeting on the biology of anticancer agents.In general, the differences in the iv) prodrugs of cis- and oxaliplatin and revealed the distinct impact of small structural modifications on their in vivo biology and anticancer activity. By this approach, we were able to identify a novel, very potent lead compound, which exerts anticancer activity superior to oxaliplatin by inducing several complete responses . Therefore, this novel drug has now been selected for further (pre)clinical development towards clinical phase I trials.Taken together, the here presented study reports the first mono-maleimide platinum(


Electronic learning (e-learning) through mobile technology represents a novel way to teach emergent otorhinolaryngology-head and neck surgery (ORL-HNS) disorders to undergraduate medical students. Whether a cognitive style of education combined with learning modules can impact learning outcomes and satisfaction in millennial medical students is unknown.The aim of this study was to assess the impact of cognitive styles and learning modules using mobile e-learning on knowledge gain, competence gain, and satisfaction for emergent ORL-HNS disorders.This randomized controlled trial included 60 undergraduate medical students who were novices in ORL-HNS at an academic teaching hospital. The cognitive style of the participants was assessed using the group embedded figures test. The students were randomly assigned (1:1) to a novel interactive multimedia (IM) group and conventional Microsoft PowerPoint show (PPS) group matched by age, sex, and cognitive style. The content for the gamified IM module was derived from and corresponded to the textbook-based learning material of the PPS module (video lectures). The participants were unblinded and used fully automated courseware containing the IM or PPS module on a 7-inch tablet for 100 min. Knowledge and competence were assessed using multiple-choice questions and multimedia situation tests, respectively. Each participant also rated their global satisfaction.P<.001) and competence . There were no significant differences in knowledge gain  and competence gain  between the IM and PPS groups. However, the IM group had a higher satisfaction score  compared with the PPS group. Using Friedman’s two-way nonparametric analysis of variance, cognitive styles  and learning modules (IM or PPS) had significant effects on both knowledge gain (both adjusted P<.001) and satisfaction (both adjusted P<.001).All of the participants  received the intended intervention after randomization. Overall, the participants had significant gains in knowledge  The large investment involved in undergraduate medical education (UME) for students and society has led medical schools worldwide to seek strategies and methods to improve their students’ progress -3. The cCertain learning characteristics have been positively correlated with academic success; for instance, strong motivation and enjoying studying have been identified as positive predictors during the undergraduate year of medical school . HoweverMobile technology has gained popularity in recent years as a means of immediate interactive multimedia (IM) communication and to access the Internet. Embedded e-learning in a smartphone or tablet can affect the educational environment. In this context, it has been termed “mobile technology in e-learning (M-TEL),” and it has been reported to represent the next natural frontier in the evolution of e-learning . Almost Reducing training time can limit the number of topics taught in an UME curriculum, including otorhinolaryngology-head and neck surgery (ORL-HNS). However, at least 20% of primary care complaints are related to ORL-HNS, and a substantial downstream effect on managing ORL-HNS problems has been reported in family medical practice . In TaiwWe conducted this prospective study from August 1, 2015 to July 31, 2017 at a university . This study included two parts: (1) pilot system-design study, and (2) validation study. This study was approved by the institutional review board of Chang Gung Medical Foundation (No: 105-5290C), and all procedures were conducted in compliance with the Declaration of Helsinki 1975. The participants were informed about the aims of the study, and written informed consent was obtained from all participants. The study proposal was registered at ClinicalTrials.gov (NCT02971735).In the pilot system-design study , we estaUsing the IM module, the learners could operate a leading character to search for and interact with other nonplayer characters to procure instructional materials, to review acquired instructional slides , and to win five small game-based quizzes . The insIn the PPS module, the learners chose and watched 10 visual-auditory text-image videos of emergent ORL-HNS disorders  by themselves. Video lectures were created by recording Microsoft PowerPoint presentations with audio narrations, timings, and ink gestures using Camtasia Studio software version 8 . The learners were free to watch the videos at any time, and they could also rewind and fast-forward the videos as needed .r=.91, P<.001, Spearman correlation test) using the Software Evaluation Checklist [During the course, all of the participants arbitrarily reviewed 80 slides by themselves. The instructional content of the two learning modules was confirmed to be correlated and equivalent by 2 investigators from the study team  age >20 years; and (2) undergraduate medical students (clerkship). The exclusion criteria were: (1) previous ORL-HNS training; and (2) declining to participate.There were four different face-to-face assessments. The cognitive style of the participants was assessed using the 25-item GEFT after enrollment . The GEFPretests including a 15-min 10-question standard MCQs to evaluate the students’ existing knowledge (range 0-100) and a 15-min 5-question MSTs to assess their existing competence (range 0-100) with regard to “emergent ORL-HNS disorders” were given to the students. Each textbook-based MCQ was designed to be answered within 90 seconds and was preselected according to the results of item analysis. The MSTs presented the learners with written descriptions of five scenarios with or without images/videos and asked them to select the appropriate responses from 5 MCQs for one emergent ORL-HNS disorder using the methodology described in a previous publication of key features approach . The MSTThe students were then asked to complete a global satisfaction score (GSS) (range 0-10) questionnaire.Blinding to the purpose of the study during recruitment was maintained to minimize preparation bias. After the participants had provided consent and completed the GEFT and pretests, we randomly assigned them (1:1) to the IM group and PPS group . A balanAfter randomization, the participants were unblinded and used fully automated courseware containing IM or PPS module on a 7-inch tablet in an ordinary office environment for 100 min. Before using the courseware, the functionality of the tablet was explained to the participants. The IM group participated in a parkour course to find and read the instructional materials and played small quiz games that were different from the MCQ and MST questions. The students in the PPS group used an app to read and listen to instructional materials in 10 linear-designed sessions. After completing the brief sessions, the IM and PPS learners could review simple slides of the instructional materials.The primary outcome measure was the percentage change in MCQ score  after the M-TEL. Other outcomes were the percentage changes in MST  and GSS.A priori sample size was estimated using primary outcome effects (percentage change in MCQ score) based on a pilot study . A two-tailed Wilcoxon signed-rank test to calculate the sample size of 26 in each group . Assuming a 10% dropout rate to fulfill the criteria of intention-to-treat analysis, we needed at least 29 participants in each group. Accordingly, we decided to enroll a total of 60 students to show the difference in percentage change in MCQ score.U test, or Kruskal-Wallis test as appropriate. Categorical variables were analyzed using Fisher exact test, and the Spearman correlation test was used to analyze the relationship between variables of interest. Friedman test  was used to compare the effect of multiple levels of two factors [P<.05. Statistical analyses were performed using G*Power 3.1.9.2 software , Statistical Package for the Social Sciences for Windows version 23.0 , and GraphPad Prism for Windows version 7.0 .Because the primary outcome measure (percentage change in MCQ score) was not normally distributed according to the D’Agostino-Pearson omnibus normality test, percentage changes  in MCQ, MST, and GSS; and AttrakDiff2 scores were compared between groups using the Wilcoxon signed-rank test, Mann-Whitney  factors . All tesA total of 60 undergraduate medical students were screened, all of whom  were randomized 1:1 to the IM group or PPS group as shown in the Consolidated Standards of Reporting Trials flow diagram (P<.001) and MST score (P=.006) after 100 min of e-learning (P<.001) and 13% , respectively. The M-TEL positively impacted the GSS (P<.001). The PPS group had significant improvements in knowledge (P<.001) and competence (P=.001), whereas the IM group had a significant improvement in knowledge (P<.001) but not in competence (P=.53). There were no significant differences in the percentage changes in the MCQ or MST score between the two groups . Notably, the IM group had a significantly higher GSS compared with the PPS group (P=.01).Overall, all of the participants showed significant improvements in MCQ score , whereas they were significantly increased in the classical FI learners . However, the percentage changes in MCQ and MST scores were not significantly different between the classical FD and FI groups . Even though the differences were not statistically significant, the classical FD learners had a lower GSS than the classical FI learners.Using the original definition of FD and FI defined by Witkin , 5 (8%) P>.05). Increases in MCQ and MST were significant in all three of these cognitive style groups. Although the differences in knowledge and competence gains were not statistically significant among the modified cognitive styles, the FINT group had a significantly higher satisfaction with M-TEL than the modified FD group regardless of which M-TEL module they used. The modified FINT learners had a significantly positive attitude toward M-TEL in terms of GSS, whereas the modified FD learners had the lowest GSS. Furthermore, the FINT learners using the IM module had a significantly higher GSS than those using the PPS module (P=.005). The modified FI learners had a positive attitude toward M-TEL in terms of GSS, and a significantly higher GSS when using the IM module compared with the PPS module (P=.02). We further compared the effect of modified cognitive style and M-TEL module on outcomes using Friedman test . Second, the study used different posttest questions to measure learning outcomes, and the interaction between taking a pretest and the intervention itself may threaten the external validity. A design which does not use a pretest would have been preferable . Third, As mentioned above, both the IM and PPS modules used the same textbook-based learning material. However, the IM module applied game-design elements and game principles in nongame contexts (gamification) . The gamTeaching and learning processes are thought to be affected by various cognitive variables. If medical students receive training, which has been designed according to their individual needs, they may develop a sense of competence and positive self-perception . PreviouMedical teachers in Taiwan have traditionally assumed that medical students can automatically adapt to the instructional modality and material by themselves to learn a topic, regardless of whether or not they are effective. Although we previously postulated that both learning module and cognitive style could affect competence gain for UME, we found the undergraduate medical students using the PPS learning module had significantly higher improvements in competence, whereas there was no change in competence in the IM learners. Even though the IM module was more attractive and satisfactory than the PPS module, more integrated formats of instructional material were needed to reduce extraneous cognitive load to facilitate problem-solving performance . BertiniSince learners can start and stop M-TEL at any time or place of their choosing , this leM-TEL using conventional PPS and novel IM modules seems to be an effective method to teach emergent ORL-HNS disorders to undergraduate medical students. The PPS module represented a formal, serious learning modality, whereas the IM module represented a satisfactory, enjoyable way for the millennial students to learn. Cognitive style and M-TEL module significantly affected knowledge gain and satisfaction, and the modified FINT learners had the highest gains in knowledge and satisfaction when using the IM module. These findings support the development of M-TEL, including various learning modules for undergraduate medical students with different cognitive styles.