Comparable, reversible hearing loss was observed for saline controls, WT phage and trans-TM peptide phage, suggesting a mild conductive hearing loss due to ME fluid. Perilymph titers after ME incubation established that few copies of trans-TM peptide phage crossed into the IE. The results suggest that, within the parameters tested, trans-TM peptides are safe and could be used as potential agents for noninvasive delivery of drugs, particles and gene therapy vectors to the ME.Otitis media (OM) is the most common infectious disease of children under six, causing more antibiotic prescriptions and surgical procedures than any other pediatric condition. By screening a bacteriophage (phage) library genetically engineered to express random peptides on their surfaces, we discovered unique peptides that actively transport phage particles across the intact tympanic membrane (TM) and into the middle ear (ME). Herein our goals were to characterize the physiochemical peptide features that may underlie trans-TM phage transport; assess morphological and functional effects of phage peptides on the ME and inner ear (IE); and determine whether peptide-bearing phage transmigrate from the ME into the IE. Incubation of five peptide-bearing phage on the TM for over 4hrs resulted in demonstrably superior transport of one peptide, in level and in exponential increase over time. This suggests a preferred peptide motif for TM active transport. Functional and structural comparisons revealed unique features of this peptide: These include a central lysine residue, isoelectric point of 0.0 at physiological pH and a hydrophobic C-terminus. When the optimal peptide was applied to the TM independent of phage, similar transport was observed, indicating that integration into phage is not required. When 10 Otitis media (OM) is a multifactorial disease with widespread consequences. It is the most common infectious disease in children aged 6 months to 6 years, and is the most common illness-related reason for which a child will visit a doctor. This leads to a significant health care burden; estimated to result in more than 5 billion dollars each year.  Out of tCurrently, the most common treatments for OM include systemic antibiotics and/or a surgical procedure involving myringotomy with insertion of a pressure-equalization tube (PET) through the tympanic membrane (TM). Systemic antibiotics frequently cause gastrointestinal and allergic side effects ,9 and haLocal pharmacotherapy is a technique successfully used in the treatment of many diseases, including otitis externa. (e.g. ) Such lo12 genetically engineered, random peptide-bearing M13 filamentous bacteriophage for the ability to cross the TM. Since nontypeable Haemophilus influenzae (NTHi) is now the most common cause of ME infection, and our goal is to provide a basis for clinically applicable improvements in pharmacotherapy, ME infection with NTHi was used during the phage selection process. Several small peptides (1000–2000 Da) were discovered that utilize an active transport mechanism to move phage particles across the intact TM. [Phage display is a discovery method stemming from a novel approach which involves screening large numbers of biomolecule species on the surface of bacteriophage.  This mettact TM.  The discThe purposes of this study were to compare the potency and physiochemical properties of five trans-TM phage particles at longer intervals than those tested previously; to determine whether an optimal trans-TM peptide phage can be identified based on transport characteristics; to determine whether trans-TM peptide can cross the membrane independent of phage; to explore the structural features of the peptides that are associated with optimal trans-TM transport; to evaluate the effects of peptide phage on the structure and function of the ME and IE; and to determine the extent to which trans-TM peptide phage may be transported into the IE.Middle Ear Response in Otitis Media (IACUC number A13-022), located at the VA San Diego Medical Center. Since phage transport takes place using an active transport mechanism, [in vivo. In order to minimize the number of animals, both ears were utilized when possible. Experimental models for OM are commonly carried out in rats due to the structural and morphological similarities between rat and human TMs. [Young adult Sprague Dawley (SD) male rats, aged 60–90 days were used for all experiments. All research was performed according to the recommendations set forth by the National Institutes of Health (NIH) in the Guide for the Care and Use of Laboratory Animals. All experiments were conducted under a protocol approved by the Institutional Animal Care and Use Committee (IACUC) titled chanism,  experimeman TMs. ,26 Anestin vivo, 48 hrs after inoculation of NTHi into the ME through the ventral bulla bone, exposed via midline neck incision, to induce OM. Care was taken to avoid the area of the TM, and TMs were visually inspected after ME inoculation to determine that no perforation was present. Five different peptide-bearing phage particles were amplified in E coli, and 109 phage particles in 50 μL of PBS were applied to one TM of six anesthetized rats per time point/peptide. After 1, 2 or 4 hrs, the external ears of the rats were rinsed repeatedly with PBS to remove any phage particles. The MEs of the rats were then isolated, and further rinsed as a precaution against any contamination. ME effusion was then harvested. The number of phage particles recovered in the fluid was titered accordingly. Lysogeny broth (LB) was inoculated with E. coli ER2738 host strain and the culture was incubated, with shaking, for 4–6 hrs. 3 mL of liquid top agar was prepared in culture tubes for each dilution and maintained at 42°C prior to use. 10 μL of each sample was serially diluted into a culture tube, vortexed briefly, and poured immediately onto plates containing LB, isopropyl beta-D-thiogalactopyranoside (IPTG), and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside . Clear blue plaques of dead bacteria on the LB/IPTG/X-gal plates were counted and multiplied by the dilution factor to convert titer results into plaque-forming units (pfu) per 10 μL. Phage titers recovered from the ME were compared to those observed for wild-type (WT) phage (not bearing a peptide) as negative controls.As noted above, utilizing phage display, we identified a number of peptides that are actively transported through the TM attached to bacteriophage.  To furthTo assess the ability of peptides to cross the TM independent of phage, TM-3 peptide was synthesized and linked to a 150 bp DNA tag for qPCR analysis . Phage was then applied to the TMs of infected rats in the same manner described above for peptide phage. 50 nmoles of the labeled peptide in 50 μL of PBS was incubated on the TM for 1 hr. The ear canal was then extensively rinsed with PBS. The ME was opened and the contents harvested and subjected to qPCR using primers specific for the DNA tag. A standard curve was generated by amplifying dilutions of the labeled peptide. This curve was used to determine the amount of peptide present in each ME.To explore the potential basis of trans-TM transport as well as differences in transport efficiency, we analyzed the structure of all five peptides. We sought to identify unique features associated with a more efficient transport rate, and with exponential increases in transport over time that would be expected with an active transport mechanism that does not saturate. To accomplish this, we evaluated the influence of amino acid composition using Pepcalc software,  amino ac9 phage particles through a well-established surgical procedure and as described previously. [The ME bullae of uninfected rats were injected with 10 μL containing 10viously. –31 FolloAfter a 24 hr ME phage incubation in uninfected rats, the animals were anesthetized and sacrificed through a cardiac perfusion. Following dissection, tissues were fixed in PFA for 24 hrs and then decalcified in 4% PFA and 8% ethylenediaminetetraacetic acid (EDTA) for 21 days. Ears were then paraffin embedded, cut into 10-μm sections, and mounted onto slides. The slides were then stained with hematoxylin and eosin so that the morphology of the ME and IE could be evaluated.To investigate possible effects of the peptides on ME and IE function, hearing sensitivity was tested prior to saline, WT phage or each of the four trans-TM phage peptides instillation into the ME, and then at 24, 48, and 72 hrs post-surgery. Pure tone ABR thresholds were obtained, using previously established methods –34 whichin vivo for 4 hrs. The IEs were harvested and extensively rinsed. An opening was made in the scala tympani wall on the cranial side of the IE. Perilymph was then sampled, first with two glass micropipette capillaries per ear, and then by micro-syringes filled with 5 μl PBS. To determine whether phage particles were present in perilymph, titering was performed as described above.To examine the possibility of TM-transmigrating phage that enter the ME might further transmigrate into the IE, rat MEs were exposed to peptides A two-way repeated measures analysis of variance (ANOVA-R) was completed using GraphPad® software. In order to investigate which means were statistically significant from one another, Tukey multiple comparisons post-hoc testing with Bonferroni correction was completed. Statistically significant was defined as differences between groups that reached a P value of <0.05. Confidence intervals were illustrated using standard error bars.The titers of five trans-TM peptide phage that were observed in the MEs of rats at 1, 2 or 4 hrs after application to the external surface of the TM are illustrated in To explore the potential basis of superior transport by peptide phage TM-3, we analyzed the structure of the five peptides, to identify unique features of TM-3 that might explain its more rapid trans-TM transport rate. We also sought to identify unique features of TM-3 and TM-4 that might explain their exponential rate of transport over time, when compared to the remaining selected peptides. Pepcalc analysis  indicateWhen the peptides were analyzed for secondary and tertiary structure in their position relative to the N-terminus of the M13 P3 protein, significant differences in predicted 3-D structures were apparent . TM-1 anWhen DNA-tagged TM-3 was applied to the TM as a free peptide for 1 hr, qPCR detected free peptide in the ME. The proportion of peptide transported was similar to the proportion observed for TM-3 phage, indicating that transport is independent of peptide linkage to phage .No significant differences were observed in ME histology when comparing the peptide phage groups to each other, or when they were compared to the two control groups. The external ear epithelium, TM, ME lumen, and ME epithelium all appeared normal and remained intact, as illustrated in ABR testing at 8, 16, and 32 kHz prior to and after ME application revealed no significant differences in the effect of any type of treatment—including all four peptide phage treatments as well as both control treatments—and no significant interactions between treatment type and day post treatment results . A signiAs seen in 9 phage in the area of the RWM, for WT, TM-1, TM-2 and TM-4. These results are compared to transit of TM-3 through the TM at the same exposure period in 6 particles recovered 4 hrs after application of 109 TM-3 phage to the TM. The figure also shows that one day after placement into the ME, from 600 to 4000 phage particles were recovered from perilymph for all phage, including WT and TM-3, approximately 106 less than the titer applied to the RWM. These results are consistent with a low level of primarily passive transit, or perhaps small amounts of contamination during the IE fluid sampling procedure.The titers of perilymph revealed that no or very few (an average of 0–67) peptide-bearing phage were observed in the IE at 4 hrs after placement of 10The existence of an active transport mechanism through the TM is surprising. The mechanisms must operate through epithelial cells of opposite polarity and through the connective tissue of the middle layer of the membrane. Whether transport occurs via the pars tensa, the pars flaccida or both is not clear. The differences between the connective tissue layer of the two parts of the TM would presumably play a limited role, since the collagen bundles that make up this layer are separated by sufficient space to allow penetration by particles the size of bacteriophage. It is clear from When the composition and structure of the five trans-TM peptides were compared, no clear conformational pattern emerged. The most consistent feature was a basic lysine or arginine in the central position. For TM-3 and TM-4, the most efficient peptides, this amino acid was at position six. However, given the many peptides in the parent library that would have shared this feature and yet were not enriched, this cannot have been a defining feature. Another feature of TM-3 not shared by any of the other peptides is the presence of 6 serine and threionine residues. With 20 amino acids, the expected average number of serine or threionine in a 12-mer peptide is 1.2 (an average diversity of 0.68 per position). However, it is not clear how the hydroxyl containing amino acids are playing a role. Similarly, when 3-D structure was compared to the efficiency of trans-TM transport of phage bearing them, no particular fold emerged. TM-3 and TM-4 showed an irregular coil structure, as did the less efficient TM-2. Meanwhile, TM-1 and TM-5 adopted an ordered helical secondary structure. The large differences in amino acid sequence composition and structure of the five trans-TM peptides could argue against a unitary ligand/receptor model for transport and a unifying single uptake mechanism. Each peptide may induce its own unique combination of molecular affects and interactions. One possibility consistent with our data is multiple binding sites that mediate a pinocytotic trans-cellular transport event. Further studies will be required to understand the mechanism(s) of peptide-mediated transport into the ME through the intact TM.TM-3 as a free peptide showed trans-TM transport that was equivalent in magnitude to TM-3 phage conjugate. This result indicates that the transport mechanism is not dependent upon attachment to phage cargo, but is determined by the TM-3 peptide itself. The fact that peptide and peptide phage transit rates were similar also implies that trans-TM transport is not influenced by the size of the transported molecular or particle. While the mechanism of active trans-TM transport is not known, the possibilities include a specific transmembrane transporter or vesicular transport within the cells of the TM, or alteration of para-cellular tight junctions allowing movement between cells.  It seemsThe hearing sensitivity of SD rats showed statistically significant threshold shifts at 24 hrs post that resolved by 72 hrs after ME application in saline controls, WT phage controls, and all 4 peptides phage (TM-1-4). These threshold shifts occurred across the frequency range tested and were comparable in magnitude for all groups. As there were no significant differences between treatments, the hearing sensitivity findings suggest a slight, presumably conductive, hearing loss due to the surgical procedure and/or the presence of the fluid injected into the ME as a carrier for the phage, or as a control. At 8 kHz, no significant difference was seen between pre-operative results compared to day 2 or day 3 thresholds, suggesting 8 kHz hearing sensitivity recovers to pre-operative status by day 2. For 16 and 32 kHz, no significant differences were seen between pre-operative thresholds and thresholds on day 3, thus sensitivity at these frequencies recovered to pre-operative status by day 3. The transient hearing loss primarily at the highest frequencies is consistent with temporary mass damping due to the fluid injected into the ME, which may have altered the impedance matching between the ear canal, ME and IE. Alternatively, tissue edema or inflammation due to surgery could have played a role in the temporary shift in hearing sensitivity. However, no visible signs of inflammation or tissue swelling were observed in sections of the ME or IE , making Our results indicate that peptide phage TM-1, TM-2, TM-3 and TM-4 are not harmful to the structure or function of both the ME and IE systems at the time points that were examined. These findings have important implications for the potential of transtympanic peptides as drug chaperones. Obviously, they can only be investigated as tools for local drug delivery to the ME in humans, once proven safe in animal studies. It is also important that minimal transit of the RWM was observed. This suggests that any off-target delivery of drugs intended for the ME would be minimal. These are critical steps in the potential therapeutic development of TM-transiting peptides.Very few TM-transiting phage entered the IE. It has been well documented that molecules can passively diffuse across the RWM during OM, including both bacterial substances such as endotoxin and host pro-inflammatory mediators.  However,With respect to peptide-mediated TM transit, if successfully developed the active transport mechanism described here could be used to deliver a wide variety of therapies to the ME. This could include small and large molecules, gene therapy vectors, antimicrobial peptides (AMPs), and particles up to the size of M13 bacteriophage .  In additIn summary, we have identified and utilized a natural cellular mechanism that mediates the active transport of large particles across the TM barrier. Several peptides with varying ability to transit the TM were identified, with rather variable sequences and predicted structures. Entry of two of these peptides increased exponentially over time, suggestive of efficient active transport. There are also no indications that peptides or phage are harmful to the external, ME or IE, or that TM-transiting peptide phage enter the IE from the ME in substantial quantities. Also, no damaging effects on hearing sensitivity were observed. The discovered peptides present a possibility for developing a local solution for ME pharmacotherapy via attachment of drugs, drug packages, gene therapy vectors or lytic phage that target OM pathogenic bacteria. This would provide a welcomed alternative to current treatment options, reducing exposure of off-target bacteria to antibiotics, side effects from systemic drug or vector delivery, as well as surgical risks. Furthermore, a noninvasive strategy for locally treating ME disease could prove to be extremely beneficial in developing countries where access to advanced medical treatment is often very limited. If simple to apply, this strategy could provide a feasible way to reduce acquired hearing loss and other serious complications due to under-treated OM. The data presented here provide evidence that this method could safely enhance local delivery of macromolecules and other therapeutics.


DDX11-AS1 play important role during HCC oncogenesis and may serve as potential therapy target for HCC.Hepatocellular Carcinoma (HCC) is one of the most fatal cancers, whose incidence and death rates are still rising. Here, we report the identification of long non-coding RNAs (IncRNAs) that associated with HCC progression and metabolism based on the systematically analysis of large scale RNA-seq data from HCC patients. We identified seven lncRNAs with high confidence which were highly related with prognostic of HCC. Of note, three of them had quite different expression patterns between the control samples and the patients, and their critical roles in cancer progression were validated. We proposed that  It was estimated that there would be over 39,000 new hepatocellular cancer (HCC) cases and over 27,000 deaths caused by it in the United States in 2016 [Long non-coding RNA (lncRNA) is a massive and diverse class of non-coding RNA genes. The lncRNAs may perform as a platform for the complicated interaction with miRNA, mRNA, protein or their complex, and they also worked as an essential regulator in almost every aspect of human body. Studies have indicated that dysregulated expression of lncRNAs had impacts on the capacities of cancer cells for tumor initiation, growth, and metastasis, which served them as promising targets for cancer diagnosis and therapy . HoweverIn this study, we studied the influence of lncRNAs on the liver cancer initiation and progression taking advantage of the large scale of RNA-seq data. We first constructed one high confidence lncRNAs expression dataset by link TCGA liver hepatocellular carcinoma dataset and a curated lncRNA databasets. Furthermore, the differentially expressed lncRNAs in the patients and their biological functions were studied.We obtained expression levels of 60483 genes from TCGA liver hepatocellular carcinoma patients and a high confidence lncRNA sets from LNCipedia [Totally, 1736 lncRNAs were differentially expressed between the control sample and the patients with a p value less than 0.01 Figure , where 4The expression levels of these differentially expression lncRNAs were plot as heatmap Figure . The samIn the center of the heatmap, the red meant the high expression and green meant the low expression as annotated in the color key. Notably, the expression levels of lncRNAs were quite stable in the control samples after normalization but were relatively heterogenic. The group of highly expressed in the control samples were annotated as light blue and that of lowly expressed as light red. Such heterogeneity of the expression levels of lncRNAs in the patients suggested the instable regulation and their important roles in cancer progression.The distribution of lncRNA of up-regulated lncRNAs and down-regulated lncRNAs across the chromosome and between positive or negative strand were plotted as Figure After p value adjustment with FDR, 7 top lncRNAs with a FDR less than 0.5 were filtered out into next analysis Table . It seemDDX11-AS1 had a significant association with survival time with a p-value of 0.0117.We also tried to fit the COXPH model to the study the relationship between the expression levels of lncRNAs and the survival status of patients. Among the 7 top lncRNAs, only However, the limited studies about these lncRNAs restricted the functional analysis. An alternative method to learn about their functions was to study the functions of their co-expressed mRNAs. Due to the large volume of potential co-expressed mRNAs, we limited our slope to the top 3 lncRNAs. The top KEGG pathways were listed in Table The curated lncRNAs were retrieved from database Lnc2Cancer  and papeThe curated lncRNAs were retrieved from database Lnc2Cancer  and papein vivo, the majority of lncRNAs did not have any functional annotation. Luckily, with the help of RNA sequencing and chromatin-state map, multiple unrelated lncRNA datasets were built independently, like LNCiepdia [In spite of the important effects of lncRNAs NCiepdia , NONCODENCiepdia , lncRNAdNCiepdia  and so fNCiepdia  naming sBesides the complex databases, there were few phenotypic information about the lncRNAs. Though a lot of common SNPs were found for the differentially expressed lncRNAs, there were no OMIM allelic variant SNPs were available till now. For example, the lncRNA HAGLROS had a lot of items in ClinVar and dbVar which suggested that it might have pathogenic effects but no real studies had studied it by experiments. More attention is needed on the study of lncRNAs.Due to the limited systematic studies about lncRNAs, we failed in finding the known function of the cancer-related lncRNAs on our top list. On the other hand, we tried to obtain the co-expressed mRNAs to figure out the possible function of these lncRNAs. Considering the large size of the available mRNA numbers even after p value adjustment, we only used the top three differentially expressed lncRNAs to learn their biological functions. The results showed that these lncRNAs were correlated with pathways in cancer, DNA repair and metabolisms Table , Table 2Though only a few studies of the differentially expressed lncRNAs on cancer were found, some side evidences were able to support their function in the liver cancer. For example, lnc-IL17RC-2 participated in T helper 17 mediated inflammatory responses, which with a large possibility was stimulated by the cancer initiation . KelemenThe survival analysis showed that the expression level of DDX11-AS1 were associated with the survival status. As a result, we would like to look more carefully at DDX11-AS1 Figure . The offExcept for this detailed study, no other study talked about the function of DDX11-AS1. As a result, though there were many SNPs or indels annotated, none of them was linked with phenotypes. There were three Copy Number Variants (CNV) appears in the region of DDX11-AS as the purple rectangle showed. These CNV had effects on this lncRNA in many aspects Table . Considelnc-C1orf222-1, HAGLROS, lnc-IGFBP7-3, lnc-FSCN1-2, DDX11-AS1, lnc-COPZ2-1 and lnc-IL17RC-2, played important roles in the liver hepatocellular carcinoma, whose dysregulated expression levels led to cancer progression. Especially, DDX11-AS1 was suggested as potential therapy targets for LHC and worth studying thoroughly.Here, we proposed that the seven lncRNAs in our top list, including th, 2016. For mRNA-Seq data, the GDC generated gene level and exon level quantification in Fragments Per Kilobase of transcript per Million mapped reads (FPKM) and provides Upper Quartile normalized FPKM (UQ-FPKM) values using GRCh38/hg38 as the reference genome. In this study, UQ-FPKM values were used as the inputs. This dataset contained 377 patient cases and 52 control samples. There were expression levels of total 60483 different coding or non-coding genes.The Cancer Genome Atlas (TCGA) datasets, including clinical information and RNA-seq results of patients with liver hepatocellular carcinoma based on Illumina Genome Analyzer RNA Sequencing, were retrieved from Genomic Data Commons (GDC) Data Portal on July 24LNCipedia is an integrated database of 118,777 human annotated lncRNA transcripts obtained from different sources , 4. TherThe expression levels of extracted 9511 lncRNAs were scale to the z-score across all the genes according to the following equation first:where μ is the mean of expression levels of each lncRNA and σ is the standard deviation. Using the scaled expression levels, R package limma  was usedFor the differentially expressed lncRNAs, we also used COXPH to study whether their expression levels were associated with the prognosis.Curated liver cancer-related lncRNAs were collected through the database Lnc2Cancer, which is a manually curated database that provides comprehensive experimentally supported association between lncRNA and human cancer . There wSince there were few studies about the functions of lncRNAs systematically, only a few lncRNAs had the functional annotations. To obtain the biological function of these lncRNAs, the co-expressed mRNAs in the liver cancer were figured out and the enriched GO terms and KEGG pathways were obtained. The correlation was calculated by linear regression and the cutoff for the coefficient and the P-value was set as 2 and 0.001, respectively . The p-v


Microfluidic-based synthesis of one-dimensional (1D) nanostructures offers tremendous advantages over bulk approaches e.g., the laminar flow, reduced sample consumption and control of self-assembly of nanostructures. In addition to the synthesis, the integration of 1D nanomaterials into microfluidic chips can enable the development of diverse functional microdevices. 1D nanomaterials have been used in applications such as catalysts, electronic instrumentation and sensors for physical parameters or chemical compounds and biomolecules and hence, can be considered as building blocks. Here, we outline and critically discuss promising strategies for microfluidic-assisted synthesis, alignment and various chemical and biochemical applications of 1D nanostructures. In particular, the use of 1D nanostructures for sensing chemical/biological compounds are reviewed. In the past several years, 1D nanoscale structures including nanowires (NWs), nanotubes (NTs) and nanofibers have drawn great attention due to their superior properties of high surface-to-volume ratio, ultrasmall scale and important applications in nanoscale device generation ,3,4. Thu−9 to 10−18 liters) of fluids, using channels with dimensions of tens to hundreds of micrometers ..11].Later, these simple designs with two or three supply channels were employed to form supramolecular structures by enabling the spontaneous self-assembly of monomers, e.g., hierarchical self-assembled β-1,3-glucan (SPG) networks b 34] an an34] anContinuous flow reactions can also be carried out using additional sheath flows to form hydrodynamic flow-focusing regime which further confines the reaction zone and avoids the direct contact of solutions with the microchannel wall. Our group introduced the microfluidic synthesis of metal-organic 1D nanomaterial on four-inlet microfluidic chips ,14. VariIn an interesting modification, fibers with controlled sizes and shapes were fabricated using hydrodynamic focusing in a microfluidic sheathing device. Straight diagonal or chevron-shaped grooves were integrated in the top and bottom walls of the flow channel to move sheath fluid completely around the core fluid, here to form poly(methylmethacrylate) (PMMA) fibers d 12]. P. P12]. PApart from the continuous flow synthesis, valve-based microfluidic systems which enable reactions in small reactors with subnanoliter volumes are also advantageous in synthesizing 1D nanostrucures. A valve-based system isolates solutions from their supplying continuous flow and provides micro-confinements where reagents can react by diffusion. For example, Tseng´s group developed such a microdevice with controlling lines and a three-electrode system  microfluidic chip was also reported by our group   technique which enabled the microfluidic synthesis of cadmium selenide (CdSe) and zinc selenide (ZnSe) structures from a solid substrate c 26]. . 26]. Since 1D nanostructures offer great potential as important building blocks for applications in various areas of nanotechnology, the controlled and predictable assembly of nanostructures to form well-ordered structures are highly desirable. Various methods relying only on external forces to align nanostructures have been reported, including electric- or magnetic field induced alignment, and dielectrophoresis . HoweverUp to now, microfluidic methods applied to assembling 1D nanostructures can be categorized as two different approaches: alignment-after-synthesis and alignment-during-synthesis techniques. The former technique involves the assembly of pre-grown nanostructures, while the latter aligning structures during the synthesis procedure.With the support from the fluidic flow and herewith, the shear forces as the guiding force, NWs and NTs can be aligned and patterned on microchips using respective pre-grown 1D nanostructure solutions. In the hierarchical assembly of NWs , the materials were synthesized first and subsequently suspended in ethanol solution . Assembl1D nanostructure can also be patterned with high coverage in confined area of microchannel by controllable reactions and flow conditions. The patterning and depositing SWNTs through the use of controlled flocculation in laminar microfluidic networks was reported . An aqueThe interaction between the two solutions at the liquid-liquid interface leads to the deposition of SWNTs on the solid surfaces  while aligning by flow direction . This teThis technique involves the simultaneous alignment of 1D nanostructures during their formation in microfluidic chips. As shown in A microfluidic device with hydrodynamically focused laminar stream was reported by Lin et al. a 16]. T. T16]. TOur group reported the microchip to trap in situ formed bundles of Cu-Asp NWs in microsized clamps, thereby enabling immobilization, positioning and cutting-out of desired lengths . As showA new and efficient method for the in situ synthesis and integration of NWs within the microfluidic device is hydrothermal reaction. As a low temperature (<100 °C) aqueous process, hydrothermal synthesis is suitable for the liquid phase reaction within the microfluidic device and holds great promise for nanostructure synthesis ,56. The In this section, it is well elucidated that microfluidic-based alignment technique is easy-to-handle and cost-effective. Thus, this approach could have great potential in using a wide range of 1D nanomaterials for the fabrication of nanostructure arrays in the future, which is useful for practical applications, e.g., single NW field effect transistor (FET) .2-coated ZnO nanorod arrays showed great selectivity, sensitivity and durability for the enrichment of phosphopeptides from tryptic protein digests . T. T69]. TThe second research effort is to develop a method for the simultaneous detection of multi-analytes. One of the first immunosensors reported was based on SiNWs and used for the detection of different antigens by SiNW-based FETs arrays . A two-cSD), the authors succeeded in chemically probing the content of numerous (~104) droplets independently and resolving the pH values and ionic strengths of the encapsulated solution. They also demonstrated the glucose oxidase (GOx) assay in droplets, measured by SiNW FET sensor and integrated optical spectrometer in parallel. Such realization of dual detection  is reported for the first time.A third research focus is the multi-readout technique e.g., electrical and optical detection on the same sensing device, which is used for verifying the sensing result and eliminating ineffective signal. A droplet microfluidic system with the compact SiNW FET which was used for in-flow electrical detection of each individual droplets was reported c 71]. B. B71]. BConductive SiNWs can also be used to probe flow velocity in microchannel. A first example was demonstrated by using SiNWs as sensitive probes for streaming potential, flow velocity and ionic strength, which also opened up the new applications of SiNWs in microfluidics . AdditioThe mostly studied nanotube is CNTs, due to the nanoscale sizes and high electrical conductivity that make them ideal substrates for fabricating electrochemical nanosensors. CNTs, the tubular analogy of graphene, can be divided into SWCNTs and MWCNTs, according to the different number of graphene sheets on the structures. SWCNTs are well known for their semi-conductive or metallic properties, depending on the specific arrangement of carbon atoms, while MWCNTs tend to behave like metals ,93. ThesIn an electrochemical sensor, typically, a three-electrode-system is involved: a working electrode based on CNTs, where the reaction takes place; a counter electrode and a reference electrode a. The naAs in the case of SiNW FETs, immunosensors based on SWCNT FETs have also been extensively investigated . The difCNTs have also been demonstrated to sense the chemical potential of solutions containing redox-active molecules ,111. TheNTs made of other materials, e.g., noble metals, show optical sensing possibilities in microfluidic systems based on surface enhanced Raman spectroscopy (SERS) . An optoThis review presents an overview of recent advances in microfluidics-based synthesis, alignment and use as sensor of various 1D nanostructures. The combination of microfluidics and micro- and nanostructures has led to significant advantages in various fields. The unique features of microfluidics, especially the laminar flow, the high surface-to-volume ratio, the enhanced mass transport and heat transfer were used very efficiently to influence reactions and control structure formation. Numerous examples proved the advantages of microfluidic synthesis vs. bulk synthesis for 1D structure formation. Although the integration of 1D nanostructures with microfluidics have achieved great progress, there are still many challenges in the synthesis, alignment and application. For example, it is still difficult to use microfluidic-assisted techniques to form diverse structures, i.e., to precisely control the morphology, dimension  or the nucleation site. In addition, the fabrication and operation of the microfluidic devices requires expertise and training. To make these approaches accessible to non-specialists, significant simplifications in the handling are required. This is particularly important since the presented 1D nanosensensors are typically not suitable for multiple uses. Cheap frames to immobilize and connect sensors and interface the nano-sized structures with micro-/millimeter connectors would be helpful in this regard.For sensing applications, multifunctional devices with high sensitivity and fast response are demanded. With respect to biological analyses or diagnostic applications where the sample is, e.g., blood and other body fluids, the major challenge is the limited specificity of the sensors. Currently, the sample must be processed before detection , or the nanostructures must be functionalized with specific molecules like antibodies to capture the analytes from a mixture. Due to the high surface to volume ratio of 1D nanostructures, a high number of antibodies or other capture molecules can be theoretically immobilized on the nanostructure, which may increase sensitivity due to the high accumulation of the analyte at the 1D structure. However, such molecules need specific conditions  and the final detection of the analyte still relies typically on fluorescence, bioluminescence or electrochemical assays and hence shows no significant improvement over surface-bound capture proteins. In other words, the increased efforts in making 1D structures is still not justified for these more complex applications. Currently, 1D structures show the most promising results when electrical/electrochemical signals are recorded, where charged molecules or polar gases are detected or pH values are measured. An exciting prospect is the possibility to transport nanowires into cells and organs where they may sense parameters like temperature or pressure, or ultimately selected molecules. Here, the uptake of the nanowires across the cell membrane as well as toxicity and biocompatibility of the material is of importance. In any case, we believe that many challenges will be solved in future and nanofibers and nanowires will become more important for sensing in ultrasmall volumes.


CNS disorders are lagging behind other indications such as oncology in implementing genotype-dependent treatment algorithms for personalized medicine. This is due to the limited knowledge about the interaction of the relevant biology and the drug’s pharmacologyWe applied a mechanism-based computer model of a cortico-striatal-thalamocortical loop of the dorsal motor circuit that has been calibrated with clinical data on antipsychotic treatment in schizophrenia patients . The Quantitative Systems Pharmacology (QSP) model is based on the appropriate connections between basal ganglia regions and consists of 220 neurons (8 different cell types), 3500 synapses and implementations of 32 CNS active targets, based on their unique locations and coupling with intracellular pathways. COMTVal156Met, 5-HTTLPR rs 23351 s/L and D2DRTaq1A1 genotypes are implemented using human imaging data in non-medicated human volunteersThe dose-dependent antipsychotic effect for risperidone, aripiprazole and paliperidone is sensitive to the COMT genotype with the MM genotype having the greatest difference with the wild-type. Interestingly the 5-HTTLPR genotype interacts with the COMT genotype: this difference is positive for 5-HTTLPRss and negative for 5-HTTLPR LL. Olanzapine, quetiapine, clozapine and haloperidol are affected much less. The D2DRTaq1A allele interacts in a complex way with the COMT genotype with haloperidol, aripiprazole and risperidone and with the 5-HTLLPR genotype for haloperidol, aripiprazole, risperidone and paliperidone. These effects are anticipated to be detectable in clinical settings.The QSP platform predicts strong and complex genotype-dependent interactions with aripiprazole, risperidone, haloperidol and paliperidone and to a much smaller degree with olanzapine, quetiapine and clozapine. These predictions could in principle be verified in clinical setting and could lead to rational personalized treatment guidance


Drosophila homolog of pheromone convertase 2 (PC2), Amontillado (Amon), as an essential protease for Slit cleavage. Further analysis indicated that Slit mobility on SDS polyacrylamide gel electrophoresis (SDS-PAGE) is slightly up-shifted in amon mutants, and its conventional cleavage into the Slit-N and Slit-C polypeptides is attenuated. Consistent with the requirement for amon to promote Slit cleavage and membrane immobilization of Slit-N, the muscle phenotype of amon mutant embryos was rescued by co-expressing a membrane-bound form of full-length Slit lacking the cleavage site and knocked into the slit locus. The identification of a novel protease component essential for Slit processing may represent an additional regulatory step in the Slit signaling pathway.Slit cleavage into N-terminal and C-terminal polypeptides is essential for restricting the range of Slit activity. Although the Slit cleavage site has been characterized previously and is evolutionally conserved, the identity of the protease that cleaves Slit remains elusive. Our previous analysis indicated that Slit cleavage is essential to immobilize the active Slit-N at the tendon cell surfaces, mediating the arrest of muscle elongation. In an attempt to identify the protease required for Slit cleavage we performed an RNAi-based assay in the ectoderm and followed the process of elongation of the lateral transverse muscles toward tendon cells. The screen led to the identification of the  Summary: The Drosophila homolog of pheromone convertase 2 (PC2), amontillado (Amon), is shown to contribute to Slit processing and further cleavage into an N-Slit, essential for Slit activity in directing muscle patterning. Drosophila embryo each muscle is formed through fusion of a single founder cell with fusion-competent myoblasts, forming a syncytium that then elongates towards its attachment sites, the ectodermal tendon cells  as an essential gene for muscle patterning. Amon is the Drosophila homolog of pheromone convertase 2 (PC2)  . It belo 2 (PC2)  and is r69B-Gal4, which drives expression before muscle elongation in the entire ectoderm. The genes tested are summarized in amon) exhibited a muscle phenotype similar to that of slit mutants when knocked down in the ectoderm. In embryos expressing amon RNAi in the ectoderm the LT muscles were closer to the segmental border  and the width of the entire segment (DS) was calculated in segments of the mutant embryos , as well as in the homozygous amon mutants. The ratio of DLT3/DS was 57% smaller than in wild type, implying that the LT muscles were significantly closer to the segmental border compared with wild-type embryos (P=0.001). Importantly, a 60% reduction in the ratio of DLT3/DS was previously reported in slit mutants  and detected . Interesamon correlated with loss of neuropeptide hormone signals from the larval ring gland and perisympathetic organs , Slit appears to accumulate in a dot-like pattern within the cytoplasm of amon mutant embryos . Representative images and quantification of the elongation of the LT muscles in amon;slit-UC-CD8 in comparison to the control of WT, amon, slit-UC, and slit-UC-CD8 alone are shown in Our previous analysis demonstrated that Slit cleavage is essential to allow anchoring of Slit-N to the tendon cell surfaces . AssociaDrosophila embryo. Its reported expression and activity in the nervous system is consistent with its ability to promote Slit processing in this system. Because knockdown of Amon in the ectoderm led to a muscle phenotype, it is assumed that Amon is expressed by the ectoderm, including the tendon cells, where it promotes Slit processing inside these cells, an activity that is required for further Slit cleavage to Slit-N and Slit-C polypeptides.In summary, the PC2 protease family member Amon is shown to promote Slit cleavage in the yw, 69b-gal4, Amon RNAi 1 and 2 (Bloomington stock no. 41635 and 28583), Sp7 RNAi (Bloomington stock no. 41612), MMP1 RNAi (Bloomington stock no. 31489), MMP2 RNAi (Bloomington stock no. 31371), amon mutant (Bloomington stock no. 29011) and slit2 were received from the Bloomington Stock Center. Slit knock-in lines were described in Antibodies used rat anti-Tropomyosin , mouse anti-Slit . Secondary fluorescent antibodies were purchased from Jackson Laboratories.Staged embryos were collected and fixed as previously described . EmbryosProtein extract was produced from staged embryos in RIPA buffer . Extract was boiled in sample buffer and run on a 7% SDS-PAGE gel, blotted onto nitrocellulose and reacted with mouse anti-Slit antibody  and HRP-conjugated anti-mouse IgG . The signal was visualized using SuperSignal (Thermo Scientific).t-test. Box plots were calculated according to Distances were measured using the line tool in ImageJ. A minimum of 5 embryos per genotype were used. Significance was calculated by


The implementation of an Internet option in an existing public health interview survey using a mixed-mode design is attractive because of lower costs and faster data availability. Additionally, mixed-mode surveys can increase response rates and improve sample composition. However, mixed-mode designs can increase the risk of measurement error (mode effects).This study aimed to determine whether the prevalence rates or mean values of self- and parent-reported health indicators for children and adolescents aged 0-17 years differ between self-administered paper-based questionnaires (SAQ-paper) and self-administered Web-based questionnaires (SAQ-Web), as well as between a single-mode control group and different mixed-mode groups.Data were collected for a methodological pilot of the third wave of the "German Health Interview and Examination Survey for Children and Adolescents". Questionnaires were completed by parents or adolescents. A population-based sample of 11,140 children and adolescents aged 0-17 years was randomly allocated to 4 survey designs—a single-mode control group with paper-and-pencil questionnaires only —and 3 mixed-mode designs, all of which offered Web-based questionnaire options. In the concurrent mixed-mode design, both questionnaires were offered at the same time ; in the sequential mixed-mode design, the SAQ-Web was sent first, followed by the paper questionnaire along with a reminder ; and in the preselect mixed-mode design, both options were offered and the respondents were asked to request the desired type of questionnaire . In total, 3468 questionnaires of parents of children aged 0-17 years  and 1194 questionnaires of adolescents aged 11-17 years  were analyzed. Sociodemographic characteristics and a broad range of health indicators for children and adolescents were compared by survey design and data collection mode by calculating predictive margins from regression models.P<.001).There were no statistically significant differences in sociodemographic characteristics or health indicators between the single-mode control group and any of the mixed-mode survey designs. Differences in sociodemographic characteristics between SAQ-Web and SAQ-paper were found. Web respondents were more likely to be male, have higher levels of education, and higher household income compared with paper respondents. After adjusting for sociodemographic characteristics, only one of the 38 analyzed health indicators showed different prevalence rates between the data collection modes, with a higher prevalence rate for lifetime alcohol consumption among the online-responding adolescents (These results suggest that mode bias is limited in health interview surveys for children and adolescents using a mixed-mode design with Web-based and paper questionnaires. The assessment of population health using health interview surveys is an established method in many countries and is a cornerstone of health reporting, health policies, and health sciences. However, epidemiological studies have shown decreasing response rates since the 1990s . The useMode effects are systematic distortions caused by different survey modes or interview situations . They ofHowever, researchers have discussed mode effects for sensitive topics. Web-based responses are associated with both anonymity and greater individualization. Consequently, SAQ-Web participants are not affected by social desirability; rather, they are less orientated toward social norms. Therefore, SAQ-Web mode yields the most honest reports, especially compared with interview modes ,14. FurtThe German Health Interview and Examination Survey for Children and Adolescents (KiGGS) is a nationally representative health interview and examination survey of children and adolescents in Germany ,19. It iWhen planning population-based  studies like KiGGS, the survey design must minimize total survey error ,25. In aAre there any differences in the prevalence rates or mean values of core public health indicators for children and adolescents aged 0-17 years between the single-mode control group using only SAQ-paper and different mixed-mode groups that combine offers of SAQ-paper and SAQ-Web?Are there any differences in prevalence rates or mean values of these indicators between the 2 data collection modes (SAQ-paper and SAQ-Web) if all online respondents are pooled and all paper-and-pencil respondents are pooled across all survey designs?The methodological pilot study used a sample of children and adolescents registered in the local resident registries of 20 municipalities in 5 federal states of Germany, covering urban and rural areas as well as the eastern and western regions of the country.Data were collected using SAQ-Web or SAQ-paper methods. All selected individuals were invited by mail to participate in the study. They were sent a cover letter with the invitation to participate, information about the study and data privacy, and an informed consent form. Depending on the allocated mode, the invitation comprised a username and password for participation through the Web option along with a paper questionnaire for those allocated to the concurrent mixed-mode design, only a paper questionnaire in the single-mode design, or only the access data for the online questionnaire in the sequential mixed-mode design. The SAQ-Web questionnaire was only optimized for desktop computers. A reminder was sent by mail to respondents who had not replied within 3 weeks of the initial invitation. Participants who did not respond to the reminder were telephoned up to 5 times 4 weeks after the initial invitation. As an additional motivation for prospective participants, each parent and adolescent who had completed a questionnaire received a shopping voucher to the value of €10. The methodological pilot study strictly adhered to the data protection regulations set out in the German Federal Data Protection Act. Participation in the study was voluntary. All parents and participating adolescents were informed about the study’s aims and content, as well as data protection, and they provided informed consent. Following the strict data privacy protocol, prospective participants between the ages of 11 and 17 years received their questionnaires only after their parents provided consent.Different questionnaires were used for different age groups. Main health indicators were included on the health questionnaires for parents of all age groups (0-17 years), and self-report data for main health indicators were obtained from adolescents aged 11-17 years. To reduce the risk of mode effects, the 2 questionnaires were designed to be as similar as possible and contained the same wording for the questions and response categories. On the basis of the unified-mode design , the worAs shown in A single-mode survey design as a control group—respondents were sent an invitation letter and paper-and-pencil questionnaires, followed by a reminder after 3 weeksA sequential mixed-mode survey design—respondents were sent an invitation letter and an online access code, followed 3 weeks later with a reminder letter and a paper-based questionnaireA concurrent mixed-mode survey design—respondents were sent an invitation letter, a paper-based questionnaire, and an online access code  andA preselect mixed-mode design—respondents were sent the invitation along with a postcard asking participants to choose one of the 2 options (SAQ-Web or SAQ-paper), followed by a reminder with the same offerThere were no statically significant differences in the (gross) sample composition across the 4 design groups in terms of known sample characteristics, such as age, sex, municipality size, region, or respondent citizenship, which were obtained from local registries.The combined response rate for all survey designs was 38.43% (n=4032), following the internationally used Standard Definitions of Outcome Rates for Surveys of the American Association for Public Opinion Research (AAPOR Response Rate 2) . There wFor this study, only survey design groups using the same version of the questionnaire were included, with 3468 completed parent-reported health questionnaires for children and adolescents aged 0-17 years and 1194 questionnaires completed by adolescents aged 11-17 years. A response was defined as one completed health questionnaire from either parents or children. Hence, a valid response did not require both parents and children to complete all requested questionnaires. To answer the first research question regarding mode equivalence across the different survey designs, we compared the single-mode control group with each of the 3 mixed-mode groups. To answer the second research question regarding mode equivalence between the 2 data collection modes, data from all survey designs were pooled .The sample compositions of participating parents and adolescents were described by various sociodemographic characteristics separately by survey design and data collection mode. The variables examined included individual adolescent characteristics ; parental characteristics ; location ; and household properties (education level and net household income). Household education level was measured using the Comparative Analysis of Social Mobility in Industrial Nations . HousehoDifferences between the control group and the different mixed-mode groups and between the 2 data collection modes were tested using chi-squared tests.A wide range of health status indicators and health behaviors for children and adolescents with high public health relevance were analyzed to identify differences between the mixed-mode designs and the single-mode control group, as well as mode differences between SAQ-paper and SAQ-Web.Lifetime diagnoses of asthma, hay fever, atopic eczema, and attention-deficit hyperactivity disorder (ADHD) were indicated by parents. Recurrent pain during the last 3 months was measured using the adolescents’ self-reports. Self-rated health (SRH) and chronic diseases were evaluated by parental report using the Minimum European Health Module questions , modifieChild and adolescent mental health problems were evaluated using the parent- and self-report Strengths and Difficulties Questionnaire (SDQ) . An SDQ As indicators of health care use, pediatrician and orthodontist visits during the past 12 months for adolescents and parent-reported visits to any doctor for children under 11 years were analyzed .t values [Health-related quality of life (HRQoL) was measured using KIDSCREEN-27 for adolescents aged 11-17 years, with 5 subscores for physical and psychological well-being, relationships with peers and parents, and school well-being. Scores were summed and transformed into t values .Adolescents reported their current smoking status, water pipe consumption during the past 12 months, second-hand smoke exposure , and lifFollowing the recommendation of the World Health Organization , healthyz or t tests.We calculated prevalence rates for dichotomous health indicators and mean values for HRQoL  by survey design and data collection mode. We compared these values using z or t tests.Due to the different sample compositions of the SAQ-paper and SAQ-Web groups (see the Results), it was necessary to control for sociodemographic characteristics to identify possible mode effects. Survey modes can differ in selection (different population groups prefer different modes) and measurement (different answers are given by the same person under different modes of administration), so these differences are confounded . AdditioFor the survey design comparison, crude as well as adjusted prevalence rates and mean values were calculated. A statistical test for diversity was conducted between the single-mode control group design and each of the 3 mixed-mode designs. Because the survey design samples did not differ in sociodemographic characteristics (see the Results) and there were only marginal differences between the 2 approaches, only the results for crude prevalence rates or mean values without adjustment for sociodemographic characteristics to simplify the presentation of results are shown here.In total, we analyzed 12 health indicators using the parental sample and 28 using the adolescent sample. For these health indicators, we tested each mixed-mode survey design against the control group. Additionally, we used 2 other statistical tests to identify differences between the data collection modes, using first the crude values and then the adjusted values.P<.02 to examine differences between the mixed-mode survey designs and the single-mode control group. For the comparison of data collection modes, a significance level of α=.05 was used.Regarding the research questions, a sensitive approach to detect possible differences  is needed. Therefore, we decided to address the statistical problem of multiple testing by correcting the significance level only for the number of tests performed for each health indicator. This was done only for tests comparing the different survey designs. We used the Bonferroni correction method to neutralize the accumulation of α-error , using aP values close to significant level are found for region of residence (P=.08), municipally size (P=.05) or child’s sex (P=.06).There were no statistically significant differences in sample composition between the mixed-mode survey designs and the single-mode control group for participating parents. However, the sample sociodemographic characteristics differed significantly between data collection modes . ParentsFor the responding children and adolescents (aged 11-17 years), there were no statistically significant differences in sociodemographic characteristics between the different survey designs, but adolescents responding online were more often male, had reached or aspired to reach higher levels of education, and were more likely to live in households with higher education and higher income, compared with adolescents who responded to the SAQ-paper .The analyzed indicators of physical and mental health status showed no statistically significant differences by survey design or data collection mode . Across No differences in the crude or adjusted prevalence rates were found in adolescent-reported 12-month use of pediatric or orthodontic services . The cruHRQoL, measured using the 5 dimensions of the KIDSCREEN-27 for adolescents, was the only indicator scale analyzed. Independent of adjustment, there were no significant differences between the 2 data collection modes (SAQ-paper and SAQ-Web) for any of the observed dimensions . RegardiThe crude prevalence of lifetime alcohol consumption (self-reported by adolescents aged 11-17 years), as well as hazardous consumption and binge drinking (based on AUDIT-C reports), showed significant differences between SAQ-paper and SAQ-Web, with higher levels of alcohol consumption reported by online participants . AlthougThere were no differences in other health behaviors assessed  by survey design or data collection mode.The main aim of this study was to examine the risk of mode effects in a mixed-mode health interview survey for children and adolescents that combined paper-and-pencil questionnaires and Web-based questionnaires. Therefore, we compared prevalence rates and mean values of a broad range of health indicators from 3 alternative mixed-mode designs  with a single-mode control group (paper-and-pencil only). We also compared results between online respondents and paper-and-pencil respondents regardless of the survey design. First, we examined differences in sociodemographic characteristics by survey design and data collection mode, as it is well documented that sociodemographic characteristics are associated with health status and health behavior ,44. RegaConsistent with previous findings, the sample composition of responding parents and of responding adolescents differed by data collection mode. We confirmed the so-called “digital divide” -50—male Prevalence rates of health complaints, such as diagnosed allergies, diagnosed ADHD, obesity, and chronic pain, were equivalent between the modes, as previous studies of adults ,11,53,58We found similar prevalence rates for SRH, chronic diseases, and impairment owing to health problems between SAQ-paper and SAQ-Web respondents. The 2 previous studies examining these health indicators among adults in general  and amonFor mental and psychosocial problems, we calculated risk groups for emotional and behavioral problems and for impairment owing to psychosocial problems based on SDQ scores . Both paIn their review of 55 studies investigating 79 instruments, Campbell et al ,62-64.All reports of health care utilization were equivalent between the self-administered modes; this is consistent with prior empirical results, including studies of adult vaccination use , adolescMost of the analyzed adolescent health behaviors  showed comparable results and no differences between the 2 modes. These results are consistent with the results of other studies on adolescents .Considering alcohol consumption, the crude and adjusted prevalence rates for lifetime consumption were significantly higher among SAQ-Web-responding adolescents. After adjusting for sociodemographic characteristics, the difference decreased but could not be explained by the sociodemographic differences between the 2 groups of respondents. The prevalence of hazardous consumption and binge drinking were comparable between data collection modes after controlling for sample composition.Most previous studies have reported no statistically significant differences in alcohol consumption among adolescents or young adults by these 2 data collection modes ,66. HoweThe higher rate of reported lifetime alcohol consumption among SAQ-Web-responding adolescents, in the absence of frequently reported hazardous consumption or binge drinking, may be interpreted in multiple ways. For example, this may be a result of different sample properties, such as SAQ-Web-preferring adolescents being more likely to experiment with alcohol consumption. However, it is also possible that this result is a mode effect based on the assumption of identical alcohol consumption in both groups. Web-based questionnaires afford greater privacy because there is no risk of parents checking the responses. Another possible explanation is the lower social orientation in the Internet mode . Both thOther empirical comparisons of measurement results between different mixed-mode survey designs are rare. In accord with one other result for the adult population , all of The strengths of the methodological pilot study are the randomized study design, the population-based sample, and the inclusion of a single-mode control group as a reference to interpret the results. However, there are also some limitations, predominantly the relatively small size of the net samples of the analyzed groups. Each survey design had a relatively low number of cases, so interpretations of the results based on the net samples must be made with caution. Possible differences across the 4 survey designs or between the 2 data collection modes could have been overlooked because of a lack of statistical power, particularly regarding the need for correction for multiple testing. Other limitations concern the external validity of the results; the study was conducted in a German setting using register-based samples of children and adolescents, so the results are difficult to generalize to other countries, settings, or populations.Our results are consistent with those of most previous studies. We found comparable results between the 2 self-administered modes (SAQ-Web and SAQ-paper) for almost all analyzed health indicators, except for lifetime consumption of alcohol among adolescents aged 11-17 years. Thus, no differences were found between the single-mode control group design and 3 mixed-mode survey designs that combined the 2 data collection modes.These results suggest that it is possible to measure health indicators for children and adolescents using a mixed-mode design combining SAQ-Web and SAQ-paper methods, with a low risk of mode effects and high comparability across different mixed-mode survey designs combining these 2 data collection modes . The imp


Gastroenteropancreatic neuroendocrine tumours (GEP-NETs) are rare cancers most often found in the gastrointestinal system or the pancreas. However, patient-reported health state utilities based on clinical trials have not been previously reported in this disease area.The CLARINET study collected EORTC QLQ-C30 data from patients in both stable and progressive disease states, although data for the latter were only available during the early stage of progression due to trial design. Using published algorithms, data were mapped to EQ-5D utility values. Random-effects generalised least squares models were used to investigate the impacts of progression status, tumour site and other patient characteristics on mapped utility values.p < 0.05) in the base case analysis, and the estimated utilities for stable and progressive disease are 0.776 and 0.726, respectively. Furthermore, scenario analyses showed that utility for progressive disease is numerically lower than for stable disease, but this may not be statistically significant in scenarios where alternative Longworth mapping algorithm was used.In total, 1053 observations from 204 patients were mapped to EQ-5D utilities using the McKenzie mapping algorithm. The final random-effects model included age, gender, baseline utility and progression status as covariates; it was not feasible to investigate time-to-death utility due to a limit number of deaths in the CLARINET study. Tumour location (midgut vs pancreas) does not seem to affect utility. However, the difference in utilities based on progression status is statistically significant  data from the CLARINET study. The decline of utility in the progressive disease state may be underestimated because progressive HRQL data were only collected shortly after the progression event in the trial. The estimated trial-based utilities can be used in future economic evaluations for GEP-NET treatments and to provide more insights to physicians on patient-reported quality of life outcomes in GEP-NETs.2005–004904-35.CLARINET EU Clinical Trials Register Number,  Gastroenteropancreatic neuroendocrine tumours (GEP-NETs) are rare tumours derived from neuroendocrine cells that can occur anywhere along the gastrointestinal tract and are comprised of a heterogeneous family of neoplasms with a wide and complex spectrum of clinical behaviour . Recent Depending on the tumour site, GEP-NETs have traditionally been divided into foregut , midgut  and hindgut tumours  . Some GEn = 101), and the placebo arm (n = 103), and the majority of patients did not have progressive disease at randomisation (195 out of 204). The follow-up period of the trial was 96 weeks. The primary endpoint was progression-free survival, and secondary endpoints included overall survival, safety and health-related quality of life (HRQL) as assessed with the European Organization for Research and Treatment of Cancer QLQ-C30 (EORTC QLQ-C30) and QLQ-GI.NET21. HRQL data were collected at randomisation and at each visit scheduled in the trial. Patients whose disease progressed were withdrawn from the trial, and one additional visit was scheduled 4 weeks after the withdrawal at which HRQL data were also collected. Therefore, HRQL data were collected for both stable disease and progressive disease in the CLARINET study, although the majority of the data were collected while patients’ diseases had not progressed.The CLARINET study is a Phase III randomised, double-blind, placebo-controlled, multinational study of the somatostatin analogue (SSA) lanreotide in patients with advanced, well-differentiated or moderately differentiated, non-functioning, somatostatin receptor-positive NETs of Grade 1 or 2 (a tumour proliferation index [on staining for the Ki-67 antigen] of <10%), with tumours originating in the pancreas, midgut or hindgut, or an unknown origin . AltogetEORTC QLQ-C30 cannot be used directly to estimate utility values. However, a number of mapping algorithms have been developed to map from EORTC QLQ-C30 data to EQ-5D-based utilities , which cUtility values, especially health state utility values, are important inputs for assessing the cost effectiveness (cost utility) of new health technologies. In a cost-effectiveness model, the time patients spent in each defined health state (i.e. life years) needs to be weighted by the utility values for that health state to estimate the quality-adjusted life years (QALYs); these are used to calculate incremental cost-effectiveness ratios  in order to determine the cost effectiveness of the new health technology. For GEP-NETs, the most common health states defined in cost-effectiveness models are stable disease (or progression free) and progressive disease; therefore, it is important that the cost-effectiveness model applies the most relevant utility values to these health states. It will also be helpful to understand how tumours of different sites (e.g. midgut versus pancreas) and time to death (e.g. the last month before death versus more than a month before death) would impact on utilities for GEP-NET patients.As far as we are aware, there is only one published study performed by Swinburn et al. that reports utility values for GEP-NET patients . In thisThe objectives of this study are to map EORTC QLQ-C30 data collected in the CLARINET study to EQ-5D-based utility values and to apply statistical models on the mapped values to estimate utility values for GEP-NET patients based on progression status (stable disease versus progressive disease), tumour site (midgut versus pancreas), time to death and other factors. The results can provide more insights on patient-reported quality of life outcomes in GEP-NETs, and the estimated utility values can be used in future economic evaluations for HTAs of GEP-NET treatments.In the CLARINET study, the EORTC QLQ-C30 questionnaire was completed by patients at randomisation [first visit] and at each subsequent scheduled visit (every 12 weeks in Year 1 [four visits] and every 24 weeks in Year 2 [two visits]) in the trial. For patients who withdrew from the study (including those who withdrew due to disease progression), additional EORTC QLQ-C30 data were also collected 4 weeks after the withdrawal in an early withdrawal visit. No further visits (and consequently HRQL data) were scheduled after this early withdrawal visit.The follow-up period of the CLARINET study was 96 weeks, and patients whose disease progressed were withdrawn from the trial. Therefore, patients who stayed in the trial for the entire trial period would have seven scheduled visits. These patients were also in the stable disease state during the trial period . For patients who withdrew from the trial, the total number of visits equals the number of scheduled visits before withdrawal plus one early withdrawal visit.If the reason for withdrawal was disease progression, then EORTC QLQ-C30 data collected at the early withdrawal visit (4 weeks after the last scheduled visit when progression was confirmed) reflects the HRQL for patients with progressive disease. It can also be argued that EORTC QLQ-C30 data collected at the last scheduled visit when progression was confirmed also reflects the HRQL for patients with progressive disease, because the actual disease progression event could occur before the visit. However, it is possible that EORTC QLQ-C30 data collected for patients who withdrew from the trial at the last scheduled visits may overestimate the true HRQL for progressive disease. This is because patients were not aware of the disease progression at these visits  and patient knowledge of disease progression could have impacted on how the EORTC QLQ-C30 questionnaire was answered. Another consideration is that the utility value for progressive disease should ideally reflect the average utility of the progressive disease health state, which can be very long for GEP-NET patients, whereas the HRQL data collected at the last scheduled visit are likely to be very close to the start of disease progression and hence not reflect the average utility for the progressive disease state. Therefore, in this analysis, the base case assumption is that only HRQL data collected at early withdrawal visits (for patients who progressed) were used for progressive disease. However, in a scenario analysis, both the early withdrawal visits and the last scheduled visits where progression was confirmed were used for progressive disease.Eight mapping algorithms linking EORTC QLQ-C30 to EQ-5D were identified from the Health Economics Research Centre mapping database –16, but The McKenzie algorithm was developed using data collected from 199 patients with inoperable oesophageal cancer treated with palliative therapies. Data were collected for both the EORTC QLQ-C30 and the EQ-5D, and the algorithm modelled EQ-5D values as a function of the EORTC QLQ-C30 global health status scale, the five function scales and the nine symptom scales . The McKThe Longworth algorithm was developed using pooled data from multiple sources across disease areas including multiple myeloma, breast cancer and lung cancer. Several approaches were attempted to develop the mapping algorithm, with the authors preferring an ordinary least squares model using nine statistically significant single-item scores as well as the age of the patient to map from EORTC QLQ C30 responses to EQ-5D values . The LonThe random-effects statistical models were fitted to the mapped EQ-5D utility values and patient characteristics collected in the CLARINET study to investigate utility values based on progression status (stable disease versus progression disease), tumour site (midgut versus pancreas) and treatment group (lanreotide versus placebo), while controlling for relevant patient characteristics such as gender, age, baseline utility  and tumour progression at baseline (progressed versus not progressed before randomisation). Time to death was not included in the statistical analysis because very few HRQL data were collected close to death (see the results section for more details).The random-effects model was deemed most appropriate in this analysis because it accounts for the nature of the HRQL data collected in the CLARINET study, specifically repeated utility observations for the same patient over time. The random-effects model accounts for this by clustering and correlation of multiple observations by patient. The random-effects model has been widely used in utility analysis based on patient-level HRQL data collected within clinical trials , 20. AllThe following model specifications were tested to inform decisions regarding the inclusion/exclusion of covariates:• Model 1: age, gender, baseline utility and progression status.• Model 2: age, gender, baseline utility, progression status and treatment group.• Model 3: age, gender, baseline utility, progression status, treatment group and primary tumour location.• Model 4: age, gender, baseline utility, progression status, treatment group, primary tumour location and tumour progression at baseline.Patients in the CLARINET study who completed at least one EORTC QLQ-C30 questionnaire were included in the analysis and their EORTC QLQ-C30 data were mapped to EQ-5D-based utilities using the McKenzie and Longworth algorithms. Table Of the 1088 EORTC QLQ-C30 assessments, 1053 (96.8%) and 1041 (95.7%) assessments were mapped to EQ-5D utility values using the McKenzie and Longworth algorithms, respectively. The unsuccessful mappings were due to the EORTC QLQ-C30 questionnaire being only partially completed and/or some patient characteristics required by the algorithms being missing. The numbers of unsuccessful mapping differ between the two algorithms because the McKenzie algorithm uses 15 domain scores of the EORTC QLQ-C30, whereas the Longworth algorithm uses nine statistically significant single-item scores from the EORTC QLQ-C30 questionnaire as well as the age of the patient.Table One aim of the analysis was to explore the impact of time to death on the utilities of patients with GEP-NETs. The estimation of time to death requires death events and the knowledge of the timing of death. Only four deaths occurred in the CLARINET study, with a further 32 deaths occurred in the CLARINET extension study  with majority of deaths occurred long after the end of the original CLARINET study. Please note that no HRQL data were collected in the CLARINET extension study and only the timing of deaths occurred in the CLARINET extension study was used in this analysis. Among the EQ-5D-based utilities mapped from EORTC QLQ-C30 in the CLARINET study, a total of 156 mapped EQ-5D-based utilities were identified that related to these 36 patients who experienced death events in either the original CLARINET study or the CLARINET extension study. Time to death for each of these EQ-5D-based utilities was calculated as the difference between the timing of death and timing the HRQL data was collected in the CLARINET study. As majority of deaths occurred in the CLARINET extension study and long after the end of the CLARINET study, majority of time to death statistics calculated were long. Specifically, there were only three EQ-5D-based utility values that were observed within 60 days prior to death (one within 30 days and two between 30 and 60 days), a further 20 EQ-5D-based utility values between 60 and 360 days prior to death, and with the rest of EQ-5D-based utility values more than 360 days prior to death. Literature suggests the impact of time to death on HRQL is important to consider, especially in the weeks preceding death , and clip-value 0.005). Age was also found to be negatively linked to utilities , whereas gender was not shown to have a significant impact on utilities .Four model specifications with different lists of covariates were tested by the random-effects models. Based on the Akaike information criterion (AIC) and the Bayesian information criterion (BIC), the simpler Model 1  had the best AIC and BIC and was therefore chosen as the base case. Table p < 0.05) than models based on the Longworth algorithm , which is consistent with the findings based on the descriptive statistics.If we focus on the impact of progression status on utility value, the estimated utility decrements across all tested model specifications were very similar when the McKenzie algorithm was applied (ranges from 0.0495 to 0.050) and when the Longworth algorithm was applied (ranges from 0.0284 to 0.0292)  were used to estimate the absolute utility values for stable disease and progressive disease based on the mean patient characteristics observed in the CLARINET study, including the mean age (62.1), mean proportion of males 52%) and mean baseline utility (0.80). The estimated base case utility values for stable disease and progressive disease were 0.776 and 0.726, respectively. Table 2% and meThe comparison shows that utility values estimated based on the Longworth algorithm are higher than values based on the McKenzie algorithm, and the estimated utility decrements are higher using the McKenzie algorithm compared to the Longworth algorithm. The utility values estimated by Swinburn et al.  and repoNo studies are presently available analysing patient-reported health state utilities in metastatic GEP-NETs. Existing literature on GEP-NETs utilities was either based on data collected from the general public  or did np-value 0.05), and the estimated utility values for stable and progressive disease using mean patient characteristics from the CLARINET study are 0.776 and 0.726, respectively. The estimated utility value for stable disease from this analysis is close to the reported utility for stable disease by Swinburn et al. (0.771) [The base case model shows that progressive disease is associated with significantly worse utility compared to stable disease ( (0.771) , which rThe estimated utility values in this analysis are also broadly in line with utilities reported in the wider population of cancer patients with similar disease progression status (i.e. progression free and post-progression for advanced stage cancers). For example, in recent NICE HTA submissions, utility values of 0.8018 and 0.7277 were reported for progression free and post-progression, respectively, for advanced melanoma , and 0.7p-value 0.05), and this strengthens the common hypothesis that patients with GEP-NETs have lower utilities in progressive disease states compared to stable disease states.The estimated utility decrement for progressive disease compared to stable disease in this analysis appears to be small (0.0495) compared to an absolute decrement of 0.159 based on the study by Swinburn et al. . There aGiven the above, this study strengthens the evidence base for HRQL and utilities experienced by GEP-NET patients and provides physicians with insights into patient-reported HRQL outcomes. The significant utility decrement for progressive disease compared to stable disease found in this analysis further supports the benefits of GEP-NET treatments with SSAs such as lanreotide that delay disease progression  due to the better HRQL for patients in stable disease states compared to progressive disease states. The utility values estimated in this analysis can be used in future economic evaluations for GEP-NET treatments, especially for patients with non-functioning midgut or pancreatic NETs of Grade 1 or 2 without disease progression.There are a number of limitations of the analysis. EQ-5D data were not collected in the CLARINET study; therefore, mapping algorithms were required to map HRQL data collected in the CLARINET study to EQ-5D-based utilities. Although mapping is widely used in economic evaluations, and in particular, many mapping algorithms between the EORTC QLQ-C30 and EQ-5D have been developed and applied in the literature, there is no specific mapping algorithm for GEP-NET patients, and the use of mapped utility values  is less than ideal and will introduce a degree of uncertainty regardless of how suitable the mapping algorithm is. In this analysis, the McKenzie algorithm used in the base case analysis tends to overestimate mild health-state utilities and, in extreme cases, to predict utilities >1. In this analysis, 12.8% of all mapped utility values generated using the McKenzie algorithm are >1 with an average utility value of 1.03 (range 1–1.06). The issue of predicting unity values > 1 has been previously reported , but theThe analysis was performed using data from the CLARINET study, which mainly recruited patients with stable disease and with well-differentiated or moderately differentiated, non-functioning GEP-NETs. Therefore, the findings and estimated utilities from this analysis is most relevant to non-functioning GEP-NETs patients whose disease has not progressed and who initially has well-differentiated or moderately differentiated disease. For patients whose disease has already progressed, the progressive disease utility value estimated in this analysis should be relevant, The results of the analysis may not be applicable to functioning patients, patients initially diagnosed with poorly differentiated disease or patients with progressive disease who have further progressed. The CLARINET study has a maximum follow-up period of 2 years for collecting health utility data. Given majority of patients in the CLARINET study were recruited during the stable phase of the disease and the slow progression nature of the disease, the relatively short follow-up period of the CLARINET trial limits the amount of health utility data collected for patients who have progressed. Another limitation is that it was not possible to carry out time to death analysis due to a lack of deaths and a small sample size of HRQL data shortly before death.Given the limitations of this analysis, it is suggested that generic HRQL instruments such as EQ-5D can be used in future GEP-NET trials to avoid the use of mapping algorithms for utility analysis. The development of mapping algorithm specific to GEP-NETs patients will also be useful to better map HRQL data collected in existing GEP-NETs trials. The collection of more HRQL data post-disease progression in future trials with longer follow-up period would also be very helpful to help estimate robust utility values for the disease progression state and time to death utilities. When such data are available, future research can estimate more robust utility values for GEP-NET patients and the results from this analysis can be compared against and validated. Another area for future research is for estimating patient-reported health utilities for functioning and poorly differentiated GEP-NET patients. Finally, future economic modelling studies can be performed to evaluate the cost-effectiveness of competing treatments for treating stable GEP-NETs patients using the estimated health utilities from this study.Patients with GEP-NETs experience worse utility values in the progressive disease state compared to the stable disease state, based on patient-reported HRQL data from the CLARINET study. The decline of utility in progressive disease may be underestimated as progressive HRQL data were only collected shortly after the progression event in the trial. The estimated trial-based utilities can be used in future economic evaluations for GEP-NET treatments and to provide more insights to physicians on patient-reported quality of life outcomes for patients with GEP-NETs.