Klinefelter Syndrome (KS) is the most common abnormality of sex chromosomes  and represents the first genetic cause of male infertility. Mechanisms leading to KS testis degeneration are still not completely defined but considered to be mainly the result of germ cells loss. In order to unravel the molecular basis of global testis dysfunction in KS patients, we performed a transcriptome analysis on testis biopsies obtained from 6 azoospermic non-mosaic KS patients and 3 control subjects.The analysis found that, compared to controls, KS patients showed the differential up- and down-expression of 656 and 247 transcripts. The large majority of the deregulated transcripts were expressed by Sertoli cells (SCs) and Leydig cells (LCs). Functional analysis of the deregulated transcripts indicated changes of genes involved in cell death, inflammatory response, lipid metabolism, steroidogenesis, blood-testis-barrier formation and maintenance, as well as spermatogenesis failure.Taken together, present data highlight the modulation of hundreds of genes in the somatic components of KS patient testis. The increased LCs steroidogenic function together with the impairment of inflammatory pathways and BTB structure, result in increased apoptosis. These findings may represent a critical roadmap for therapeutic intervention and prevention of KS-related testis failure.The online version of this article (doi:10.1186/s12864-015-1356-0) contains supplementary material, which is available to authorized users. Klinefelter Syndrome (KS) is the most common abnormality of sex chromosomes, with a prevalence of 1:600-1:1000 in newborn males, even if this condition is generally diagnosed in the adulthood . KS subjThe study was approved by the local Ethics Committee of the University Hospital of Padova and was in accordance with the Helsinki II Declaration. All participants were asked for and provided their informed consent. Bilateral testicular biopsies were obtained from 6 azoospermic non-mosaic KS patients (mean age 28.4 ± 3.7) affected by Sertoli cell-only syndrome and from 3 controls subjects (mean age 29.1 ± 4.4) with obstructive azoospermia and normal spermatogenesis. Details of method and processing of testicular biopsies are described in our previous study .The data sets supporting the results of this article are available in the GEO public repository database, [ID: GSE54023;


A preparation method for quantification of bacteria in tissues is obligatory to reduce tissue mass, concentrate the target, purify, remove inhibitory substances and to achieve constant target recovery rates. No preparation method has been available until now for a high mass of tissue applicable for routine use and analytical veterinary diagnostics.Listeria monocytogenes as model organism, viable cell quantification or DNA extraction and quantitative real-time PCR were performed after Matrix-Lysis to determine recovery rates and detection limit (LOD). The adapted Matrix-Lysis protocol resulted in high recovery rates  for all tested organs, except kidney, and recovery was constant over 5 log scales for all tested buffer systems. The LOD for Matrix-Lysis with subsequent plate count method (PCM) was as low as 1 CFU/5 g, while for qPCR based detection the LOD was 102 bacterial cell equivalents (BCE)/5 g for two buffer systems.This study describes an easy-to-use tissue preparation protocol to quantify Gram-positive bacteria from a large volume of tissue matrix. A previously published sample preparation method (Matrix-Lysis) from food science was successfully adapted for clinical use on tissues from pigs, including cerebrum, spinal cord, lung, liver, ileum, colon, caecum, kidney and muscle tissue. This tissue preparation method now permits quantification of pathogens from 5 g of organic matrix, which is a 20–200 fold increase by weight compared to other methods. It is based on solubilization of the sample matrix with either a chaotrope plus detergent or divalent salts as solubilization agents. The method was designed as a modular system, offering the possibility to change lysis buffers, according to tissue solubilization characteristics and the intended detection method (molecular or culture). Using This tissue preparation is inexpensive and can be easily used for routine and analytical veterinary diagnostics. Inoculation studies or hazard assessments can profit from this tissue preparation method and it is anticipated that this study will be a valuable source for further research on tissue preparation strategies. Molecular tools for detecting, analyzing and quantifying specific microorganisms are well established in modern analytical diagnostics. The rising demand for fast quantification tools for pathogens has led to a tremendous increase in methodological approaches focusing on molecular biological techniques, such as quantitative real-time PCR (qPCR) -3. Real-PCR assays have been published for almost all known bacterial pathogens, targeting resistance or virulence genes or conserved regions ,6. HowevBesides qPCR, other methods can benefit from the abilities of a tissue preparation method to reduce the tissue matrix and concentrate target bacteria. For example, plating microorganisms is a common approach to qualitative pathogen detection, but where enrichment steps are necessary prior to detection, quantification becomes impossible. A high background flora on plates might strongly influence the growth of target organisms. This, as well as the methodological problem of low-occurrence of pathogens, is frequently discussed in the literature -14. A diIn general, pathogen quantification is always a multistep process, which is also referred to as an analytical chain. This chain can be subdivided into links comprising “sampling, tissue preparation, and plating” or “sampling, tissue preparation, DNA extraction/isolation, and target amplification/detection” . Each inImplementation of a cost-effective, robust and easy-to-use tissue preparation method prior to pathogen detection remains challenging in clinical applications, and is still problematic in nucleic acid testing . Until nIn food microbiology, requirements for a food sample preparation method are now well defined and can be specified as follows: i) reduction of matrix size for downstream steps without loss of target bacteria, ii) concentration of the target, iii) purification and removal of inhibitory substances, and iv) a low detection limit and constant recovery rate of the target . Many saHowever, a promising sample preparation method that can overcome these problems is Matrix-Lysis, which was initially developed in food science for application with dairy and meat products . The MatListeria (L.) monocytogenes was used as a model Gram-positive organism.The aim of this study was therefore to apply and adapt the Matrix-Lysis food sample preparation method to different healthy organs and to introduce it as a tissue preparation method for routine use and analytical veterinary diagnostics. The end objective was to achieve a detection limit of <100 BCE /g, which is claimed to be required urgently in diagnostics . A metho2 with optional 1% Lutensol AO-07) on clinical tissues. The solubilization capability was considered sufficient if the remaining pellet after Matrix-Lysis was small enough for downstream analysis. The threshold for the pellet size was set at <350 mg. This corresponded to a pellet mass of 7% compared to the mass utilized (5 g). A remaining pellet size >7% was considered insufficiently solubilized. Six out of nine clinical tissues were solubilized sufficiently using the 8 M urea + 1% SDS lysis buffer. Insufficient lysis was achieved with lung, ileum and caecum. Only three sufficient tissue lysates were obtained using the 2 M MgCl2 lysis buffer. Liver, colon, kidney, lung, ileum and muscle tissue produced pellets more than 7% volume. The resultant pellet size from five tissues was considered sufficient with the 1% Lutensol AO-07 added to the 2 M MgCl2 lysis buffer. Liver, lung, ileum and caecum could not be lysed sufficiently. Details about the remaining pellet sizes are listed in Table The Matrix-Lysis protocol was applied as published , for tes2 lysis buffer was insufficient for lung and muscle tissues. However, all tissues were solubilized sufficiently using the 2 M MgCl2 + 1% Lutensol AO-07 lysis buffer (Table Matrix-Lysis was repeated making use of the adapted protocol (this study) to achieve more efficient tissue lysis. An additional homogenization step after incubation in lysis buffer was introduced and pre-warmed buffers  further enhanced the solubilization process. The complete protocol established for clinical tissue is shown in Figure 2 + 1% Lutensol, was used and subsequently DNA extraction and qPCR were performed for quantification. The tissues were spiked with approximately 105 BCE/g. Overall recovery after Matrix-Lysis was within 1 log10 unit for all nine tissues. Unexpectedly, kidney tissue showed consistently lower recovery rates (15% ± 7.9%) compared with other organs. Mean recovery rate of target cells from organs, excluding kidney, was 76% ± 39%. To test the stability of recovery rates through dilution series, extended artificial contamination experiments over a five log10 dilution series were performed with 5 g of cerebral tissue. Lysis buffers used for Matrix-Lysis were 8 M urea + 1% SDS and 2 M MgCl2 + 1% Lutensol and qPCR was used for quantification. The amount of inocula ranged from 102-106 BCE/g tissue. With 8 M urea + 1% SDS buffer for Matrix-Lysis, mean recovery rate was 32.5% (R2 = 0.99). When the buffer was based on 2 M MgCl2 + 1% Lutensol, mean recovery was 104.3% (R2 = 0.99).Results of artificial contamination experiments for nine different tissues are presented in Figure 2 lysis buffer-treated samples, since 2 M MgCl2 is known to preserve the viability of bacteria and this makes a plating approach possible. The amount of inocula ranged from 101- 105 CFU/g. The mean recovery rate for L. monocytogenes using Matrix-Lysis, with a lysis buffer based on 2 M MgCl2, was 96.1% (R2 = 0.99) after PCM.The PCM method (ALOA agar) was used for quantification of the 2 M MgClThe major advantage of Matrix-Lysis tissue preparation lies in its efficiency and robustness to recover and concentrate as few as one bacterial cell from a large sample mass (5 g) into a pellet small enough for subsequent molecular and microbiological detection methods. To determine if tissue preparation with Matrix-Lysis is as efficient at low bacteria numbers as it is with higher numbers, tests were performed to determine both the LOD and reproducibility of the Matrix-Lysis tissue preparation method. This was performed by testing triplicates of low bacteria counts  revealed excellent stability of the method, independent of the target quantity. This offers new and promising strategies for quantitative plating approaches in analytical veterinary diagnostics. Matrix-Lysis, followed by quantification with qPCR, can produce the same mean recovery rates as published before using Matrix-Lysis for food matrices [The constant mean recovery rate over the dilution series after PCM . For the lysis buffers based on 8 M urea + 1% SDS and 2 M MgCl2 + 1% Lutensol, constant recovery down to 349 BCE/g and 406 BCE/g, respectively, could be achieved with qPCR. As shown in this study, the newly developed Matrix-Lysis tissue preparation method is capable of recovering and concentrating single bacterial cells from as much as 5 g of tissue sample, efficiently and robustly.Spiking experiments with cerebrum over the whole dilution series resulted in a very high coefficient of determination  of the sample eluate after DNA-extraction, which inevitably increases the overall detection limit [Nevertheless, the discrepancy between the LOD of one cell with PCM compared to that of 10on limit . Secondlon limit . Finallyon limit , the colAdditional file If only qualitative results are required, novel approaches, such as molecular enrichment, can circumvent these limitations  and thisAs previously indicated, DNA-extraction is an important bottleneck step for qPCR-based quantification and from the results of this study we suggest the application of internal cellular-based extraction controls, to control for inefficient extraction and purification . NeverthIn summary, it can be concluded that the Matrix-Lysis protocol from food science can be successfully adapted to clinical tissues. To our knowledge this is the first study presenting an easy-to-use and economical tissue preparation method for routine use and analytical veterinary diagnostics. The tissue preparation method permits quantification of pathogens from 5 g samples of organic matrix, which is 20–200 fold higher compared to prior sample preparation methods. Following Matrix-Lysis, the remaining pellet contains the total bacterial population of the sample. This pellet can then be further processed with DNA-based quantification or plating methods. It can be used for clinical research and diagnostic monitoring. For example, enumeration of lactobacilli and other Gram-positives can be useful for studying effects of dietary manipulations in pigs  or pathoCerebrum, spinal cord, lung, liver, ileum, colon, caecum, kidney and muscle tissues were obtained from slaughtered pig carcasses from a slaughterhouse in lower Austria and from tissue provided from the Clinic for Swine, University of Veterinary Medicine, Vienna. No animals were euthanized for the purposes of this research. Exact anatomic sampling sites are listed in Table L. monocytogenes  was conserved at −80°C using MicroBank technology  and constituted part of the collection of bacterial strains kept by the Institute of Milk Hygiene, University of Veterinary Medicine, Vienna. L. monocytogenes was incubated overnight (37°C) in tryptone soy broth (TSB) with 0.6% (w/v) yeast extract .1- 106 BCE or respectively CFU. For the spiking trial of nine different tissues, a target concentration of approximately 105 BCE/g was used. Ice-cold tissues were cut into small pieces (Ø <4 mm2) and spiked with pipetted droplets of culture of known concentration. Tissues were processed immediately. All spiking experiments and determination of the LOD were performed in triplicate. All organs used for artificial contamination experiments were tested to be negative for L. monocytogenes prior to inoculation using the Matrix-Lysis protocol and the real-time PCR assay described by Rossmanith et al. [For artificial contamination of tissues a 1 ml aliquot was taken from the overnight culture, placed into fresh TSB and directly incubated at 37°C for another three hours. Subsequently, a ten-fold dilution series was prepared using phosphate buffered saline (PBS) as diluent. The amount of inocula (100 μl) for spiking dilution series using cerebral tissue ranged from approximately 10h et al. .All spiking experiments were controlled by three methods: using the plate count method (PCM), microscopic enumeration and a qPCR step to check for possible discrepancies between the different enumeration techniques. Tryptone soy agar plates, supplemented with 0.6% (w/v) yeast extract , were used for the PCM. Dilution series of the cultures were plated and incubated at 37°C for 24 hours. Microscopic investigation was performed with 500 μl of an appropriate dilution of bacteria, following the protocol provided in the Live/Dead® BacLight kit . Samples were analyzed using a Laborlux 8 fluorescence microscope  with a 470 nm filter and a 1,000-fold optical magnification. Two filters were analyzed per sample and fifteen visual fields were counted per filter. The following formula was used for calculation of the number of stained bacteria per milliliter: mean bacterial count in one field X (filtration area size/field size) X (1/dilution factor × 0.5 ml).2, 50 mM Tricine and optionally 1% Lutensol AO-07 and a pH adjusted to 7.0 . All reagents for the Matrix-Lysis protocol were purchased from Merck , with the exception of SDS, Tricine , Lutensol AO-07  and Savinase .The solubilizing ability of different buffer systems was examined for testing the applicability of Matrix-Lysis on clinical tissues. Matrix-Lysis buffers  were prepared as previously described . Matrix-2 lysis buffers, with and without 1% Lutensol. The Matrix-Lysis protocol previously published [2) and homogenized with the Stomacher 400 blender  in 25 ml of sucrose-protease buffer for ten minutes. The homogenate was transferred to a sterile 50 ml polypropylene tube  and incubated in a water bath at 37°C shaken at 250 rpm for 30 minutes. Samples were then centrifuged at 3,220 × g at room temperature for 30 minutes. The supernatant was decanted and the pellet resuspended in 30 ml lysis buffer. Samples were then incubated in a water bath at 37°C (lysis buffer based on MgCl2) or 42°C (lysis buffer based on urea) and shaken at 250 rpm. Subsequently, samples were re-homogenized for ten minutes. Resultant sample homogenates were then made up to 45 ml with lysis buffer and vortexed rigorously. Diluted homogenates were centrifuged at 3,220 × g for 30 minutes at room temperature. Supernatants were decanted and pellets resuspended in 40 ml of washing buffer. Samples were then incubated in a water bath for 30 minutes (shaken at 250 rpm), at the same temperature used during incubation in lysis buffer. Incubated samples were next centrifuged at 3,220 × g for 30 minutes at room temperature and the supernatant decanted. Pellets were resuspended in 1,500 μl PBS and transferred to a 2 ml plastic tube . Pellets were washed twice using 1,500 μl PBS in centrifugation steps . The adapted protocol is shown in Figure In this study the Matrix-Lysis protocol was tested using the lysis buffer 8 M urea + 1% SDS, and both variants of MgClublished  was follL. monocytogenes in clinical tissues after Matrix-Lysis, the remaining pellet was either plated onto selective agar plates for viable cell quantification or DNA extraction was performed for subsequent qPCR enumeration. The PCM was used for viable cell quantification. The remaining pellet from Matrix-Lysis was resuspended in PBS to achieve a total volume of one ml and rigorously vortexed until homogenized. 100 μl aliquots of a ten-fold dilution series were plated onto selective ALOA agar . DNA extraction was performed with the NucleoSpin® tissue kit  using the protocol for Gram-positive bacteria.For quantification of L. monocytogenes was performed by targeting a fragment of the prfA gene (274 bp) [L. monocytogenes DNA and a derivative 4 log10 dilution series were used as calibration standards for each run. The qPCR reaction on 25 μl samples was performed in a Mx3000p qCR termocycler  in duplicate using 5 μl of DNA template.Since somatic cells remain in the Matrix-Lysis pellet and result in high background DNA levels and overload of the DNA extraction column, only 1/2 of the B3 buffer lysate was used for further DNA extraction. Following extraction, qPCR quantification of (274 bp) ,33. A DNhttp://biomedcentral.com).Supplementary Information accompanies the paper on the BMC Veterinary Research Journal website  their work.EM performed the experimental work, analysed the results and composed the manuscript. KP and PM participated with the spiking experiments and real-time PCRs. MW participated in the design of the study. PM and PR helped with design and coordination of this study and in editing the manuscript. All authors read and approved the final manuscript.Influence of background DNA concentration on DNA extraction efficiency. 30–300 μg of salmon sperm-DNA was added to the DNA extraction system to determine changes in quantification efficiency in the presence of background DNA.Click here for file


In 2010, 7.6 million children under five died globally – largely due to preventable diseases. Majority of these deaths occurred in sub–Saharan Africa. As a strategy to reduce child mortality, the Government of Malawi, in 2008, initiated integrated community case management allowing health surveillance assistants (HSAs) to treat sick children in communities. Malawi however, faces health infrastructure challenges, including weak supply chain systems leading to low product availability. A baseline assessment conducted in 2010 identified data visibility, transport and motivation of HSAs as challenges to continuous product availability. The project designed a mHealth tool as part of two interventions to address these challenges.A mobile health  technology – cStock, for reporting on community stock data – was designed and implemented as an integral component of Enhanced Management (EM) and Efficient Product Transport (EPT) interventions. We developed a feasibility and acceptability framework to evaluate the effectiveness and predict the likelihood of scalability and ownership of the interventions. Mixed methods were used to conduct baseline and follow up assessments in May 2010 and February 2013, respectively. Routine monitoring data on community stock level reports, from cStock, were used to analyze supply chain performance over 18–month period in the intervention groups.Mean stock reporting rate by HSAs was 94% in EM group (n = 393) and 79% in EPT group (n = 253); mean reporting completeness was 85% and 65%, respectively. Lead time for HSA drug resupply over the 18–month period was, on average, 12.8 days in EM and 26.4 days in EPT, and mean stock out rate for 6 tracer products was significantly lower in EM compared to EPT group.Results demonstrate that cStock was feasible and acceptable to test users in Malawi, and that based on comparison with the EPT group, the team component of the EM group was an essential pairing with cStock to achieve the best possible supply chain performance and supply reliability. Establishing multi–level teams serves to connect HSAs with decision makers at higher levels of the health system, align objectives, clarify roles and promote trust and collaboration, thereby promoting country ownership and scalability of a cStock–like system. In 2010, 7.6 million children under five died globally – largely due to preventable diseases including pneumonia 14%), diarrhea (10%), and malaria (7%) . iCCM in%, diarrhIn 2008, the Government of Malawi (GoM) initiated iCCM as a strategy to reduce child mortality. The program entailed training an existing cadre of CHWs, known as Health Surveillance Assistants (HSAs) to treat children in the community. HSAs are posted nationwide to serve communities at a ratio of 1:1000 population. By September 2011, 3296 HSAs had been trained in integrated management of childhood illness (IMCI) . With thDespite the gains made towards MDG 4, the health care infrastructure in Malawi still faces challenges, among them weak supply chain systems. This affects continuous health product availability at the community level, consequently undermining the full effectiveness of the iCCM strategy. In order to identify the constraints associated with maintaining regular product availability, Supply Chains for Community Case Management (SC4CCM) in collaboration with the Ministry of Health (MOH), conducted a formative assessment across 10 districts in Malawi  in 2010 . The assTo address the identified constraints related to data visibility, motivation and transport, SC4CCM designed and piloted cStock, a mHealth tool for community–level reporting of stock on hand data and resupply of 19 health products managed by HSAs. cStock was nested within two broader interventions, namely, Enhanced Management (EM) and Efficient Product Transport (EPT), to address challenges in motivation of HSAs and transport to the health facilities, respectively. The primary objective of this paper is to assess the feasibility, acceptability, and effectiveness of cStock as a mHealth strategy for improving data visibility and reducing stockouts of health products used at the community level. Additionally, the study will explore the added effects of the team approach deployed through EM in improving supply chain performance.cStock is an SMS and web–based reporting and resupply system that is used by HSAs to report stock data via SMS through their personal mobile phones. cStock calculates HSA resupply quantities and sends this information to HF staff to use to pick and pack products for HSAs and notify them about a collection time.cStock was designed using a consultative, user–centered and iterative process. Potential users at all levels of the health system provided inputs based on their experience with the existing manual reporting and resupply system. Information on the existing flow of data across levels of the health system was combined with inputs from supply chain specialists to ensure the system was based on supply chain best practices. In designing the workflows and dashboard for cStock, an important criterion was to ensure the health care workers and managers at each level would have access to data most relevant to them, at the right time and in a format that could be easily accessed, interpreted and used for decision making. HSAs and HF staff would interact with cStock using SMS messages on their own phones, while district and central level staff would receive alert messages from cStock on their own phones. District and central level staff would use computers to access the web–based dashboard for reports. The dashboard was redesigned six months after implementation so that district and central level users could incorporate their experience interacting with the system into the redesign and prioritize metrics and visuals most useful for their day–to–day operations.cStock is a key component of both the EM and EPT intervention packages. The EM intervention addresses challenges related to data availability and visibility, as well as low motivation among HSAs while the EPT intervention addresses challenges of transport in addition to data visibility. The additional component of the EM intervention was District Product Availability Teams (DPATs). These are multilevel quality improvement teams that use data supplied by cStock to monitor performance of the supply chain and make informed supply chain decisions. In contrast, the additional component of the EPT intervention consisted of training all HSAs on bicycle maintenance, provision of a basic tool kit, and the use of a continuous review inventory control system.HSAs and HF staff in six districts where the project was piloted were trained on the use of cStock for reporting and resupply and used their own phones to register with cStock. Training was conducted using a cascade approach. The project trained a group of trainers consisting of central and district level staff from pilot districts as TOTs and they in turn trained the HSAs, HF and district staff. Both intervention groups were trained over a 2–day period, with one day dedicated to training users on how to register and use cStock. HSAs in the EM group were trained to send reports to cStock using a fixed monthly reporting schedule between the last day of the month and second day of the next month, while those in the EPT group were trained to send reports to cStock at any time during the month when they planned to travel to the HF giving them the flexibility to report and receive product. Once trained, HSAs were provided with job aids to carry back to their facilities and expected to start using cStock immediately. Implementation support for cStock was provided through group messages sent by the system administrator to users to correct common errors, automated messages sent directly by the system to users in response to formatting errors, and field visits made by Ministry and project staff to monitor and reinforce good practices.In designing the interventions, the project identified feasibility and acceptability of cStock as critical components to enhancing uptake and laying the foundation for country ownership and scalability. See 2 and shares boundaries with Zambia in the west, Mozambique in the east, south and southwest, and Tanzania in the north. The country is divided into three administrative regions, namely the northern, central and southern regions with 28 districts in total  is also good because we are able to send the messages at any time even in the night and we get the response in time.”FGD findings highlight the relevance to users include HSAs description of cStock and its components as a fast system for sending messages and receiving feedback. An HSA Supervisor in the EPT group offered: “Yes, [cStock is] very useful because communication is very fast between the HSA and the In–Charge, whenever they want emergency order they receive feedback right away from the health facility.”FGD findings related to the theme of … [cStock] is a linkage between the HSAs and the supervisors, even the In–Charge because when the products are ready we just send the message to let them know that the drugs are ready. While before it was hard to reach every HSA.” Users also noted that the cStock system helps users remember to take necessary actions, for example one HSA in the EPT group stated, “cStock is good because it reminds you what to do, like if you have one day left to send the report it always reminds you that you have one day left.”HSAs and HF staff also perceived cStock as a linkage that facilitates communication between levels from the villages to the HSAs, HSAs to the HF. One HSA Supervisor in the EPT group offered, “routine use, its effect on users’ daily work, and perceived benefits identified by the user. Data for the six intervention districts, analyzed over 18 months from Jan 2102 – June 2013, showed a steady increase (79% to 99% in EM and 71% to 90% in EPT) in routine use of cStock to report on stock levels averaging at 94% for EM, 79% for EPT. Follow up findings also showed that a large majority (97% in EM and 91% in EPT) of HSAs reported that cStock had become their primary means for ordering or requesting health products from their resupply point. Additionally, 92% of health facility Drug Store In–Charges in EM and 91% in EPT reported using cStock to determine the quantities to resupply to HSAs. The acceptability of cStock was evaluated by looking at its level of In addition to routine orders, HSAs also reported using cStock as the primary means for submitting emergency orders. While at baseline, 52% of HSAs across six districts (n = 98) reported travelling to the HFs to submit emergency orders, this percentage dropped to 28% in EM and 21% in EPT at follow up, with the majority  of HSAs now using cStock to send emergency orders. All HSAs  also reported using cStock routinely to send receipt messages upon collecting their products. Most (96% in EM and 87% in EPT) of the HF staff interviewed reported always using cStock to inform HSAs of orders ready for collection; 44% of HF staff in EM and 30% in EPT used it for follow–up on non– and incomplete reporting, both critical SC performance indicators, particularly for the DPATs.There is more transparency now as drugs are not given on a friendly basis, when you find products are ready, we count with the specific HSA for accountability...”FGD findings show that cStock contributed to improved accountability for managing products, as HSAs, HFs, and district staff were all responsible for and played roles in reporting and communicating stock–related issues. One positive result of this is reduced product wastage; an HSA Supervisor from the EM group explained “l feel very happy whenever l am sending SMS to cStock because l receive feedback right away and because of this I am motivated.” An HSA Supervisor from EPT adds, “Yes, it’s very useful because communication is very fast between the HSA and the In–Charge, whenever they want emergency order they receive feedback right away from the health facility.” cStock provides useful information for monitoring SC performance. HF staff reported that cStock enables supervisors to monitor HSA performance and supervisors’ own performance through self–assessment. In the EM group, a HSA Supervisor offered “…we look at their reporting rate, the time they sent their report, the completeness of the report and supervisor notes that, on cStock helps us to monitor performance as the information is easily available.”According to FGD participants, cStock is an important source of feedback and information. HSAs perceive monthly cStock reminders about reporting as useful, and HF staff noted that cStock alerts are beneficial in notifying them of late reporting from HSAs. An HSA in the EPT group shared “as my friends have said, transport costs have been reduced significantly and that is good for us.” HF staff reported being able to manage inflow of HSAs to the HF to collect products by picking and packing in advance as well as sending notifications to HSAs that their order is ready thus serving them better. cStock also increases staff ability to monitor and communicate information on product usage. HSAs reported that cStock allows tracking product distribution and use, both at the HSA Supervisor level and higher. An HSA from the EPT group added, “… there is transparency on cStock that your boss is able to know if the products are distributed.”FGD participants felt that cStock increases the overall efficiency of the community supply chain system. HSAs reported that cStock has resulted in a marked reduction in the significant effort, time, and/or money they had previously spent in collecting products from the HF. One HSA in the EM group shared, “Acceptability of the DPAT component of the EM intervention was evaluated by looking at how districts, HF staff and HSAs used information supplied by cStock for routine monitoring and managing of community SC performance, improving on their DPAT targets and rewarding well performing HSAs. All district staff in EM districts reported visiting the dashboard at least once a week to obtain data from cStock for planning, coordination, and supervision. District staff also printed cStock reports for use by HF staff to monitor HF and HSA performance. HF staff reported using cStock information obtained either through the resupply worksheets or cStock reports to discuss reporting (79%) and stock management (53%) during DPAT meetings. Seventy–eight percent of the HSAs said their supervisors referred to the performance targets such as reporting during the DPAT meetings and 80% knew they would receive rewards if they performed well in stock management using cStock. sometimes it happens that you receive less products when you see more cases, we discuss how best to cater for these cases.” HSA Supervisors reported discussing similar topics, such as HSA performance, supply management, stock outs, reporting rates, and recognition of good performance among HSAs. And HSAs described being motivated by the performance monitoring, positive feedback, and performance targets used as part of the DPAT design. Another HSA in the EM group stated, “for us we share ideas during the meeting, as a single person you cannot build a house, we are motivated.” HF staff also cited DPAT meetings as useful in providing a forum for reviewing SC issues and performance.One aspect that made EM acceptable to users was the DPAT role in monitoring SC performance. HSAs reported that DPAT meetings were useful for discussing SC issues, such as HSA performance, reporting rates, and performance targets. One example from an HSA in the EM group is “HSAs and HF staff said that EM helped link people and activities across the supply chain, aligning the flow of information and clarity of processes. Challenges to DPATs included HSA attendance, HSA hunger when no food or refreshments were offered, lack of follow up on issues discussed at meetings, lack of needed materials, and poor coordination/communication by HF In–Charge.In terms of comparison with the EPT group, performance on most acceptability indicators in Findings on effectiveness are presented as a comparison of supply chain performance between the EM and EPT groups according to these four indicators: reporting, complete reporting, lead time, and stock out rates over time, using data from cStock dashboard reports.t(106) = 6.9766, P < 0.001  compared to the EPT (79%) group. There was a slow increase in time for both groups, and greater variation seen within the EPT group than EM. This study found that the EM group had significantly higher reporting rates (94.0 ± 10.7%) compared to the EPT group (79.1 ± 11.0%), t(106) = 9.8953, P < 0.001.Completeness in reporting measures the extent to which HSAs send in stock on hand messages to cStock for all the products they manage. Mean completeness rates were found to be higher among EM (85%) than EPT districts (65%). Lead times. On average, lead times, a measure of responsiveness in the supply chain, were significantly (P < 0.05) lower/better in EM compared to EPT districts with HFs in the EM group taking on average 12.8 days to fulfill an HSA order compared to 26 days in the EPT group. The difference in the two groups was small (four percentage points) in Jan 2012, increasing (to 24 percentage points) in June 2013. This study found statistically significantly lower lead times (12.8 ± 6.2) in the EM group compared to the EPT group (26.4 ± 11.3), t(106) = –7.75, P < 0.001  and sustained as shown by the data extracted from cStock over 18 months. The study also found that following training, HSAs readily adopted and routinely used cStock as an alternative method for ordering products on their own volition without institutional strength ordering them to do so. This rapid uptake of cStock occurred even with the existence of a government–approved paper system that served the same function and can be attributed to the immediate perceived benefits that included a communication and feedback loop related to product requests. The high cStock reporting rates sustained over time despite the presence of an alternative mechanism for sending reports are a further indication of user preference and acceptability of the system. Additionally, empirical evidence from the cStock dashboard shows that HSAs continued using cStock after the pilot at nearly the same or similar usage rates at the follow up assessment.The difference noted in reporting rates across the two study groups demonstrates the added benefit of DPATs – teams created in EM districts to enhance data utilization and reward HSAs for good performance of SC tasks. While feasibility and acceptability were equally high in both groups, the enhanced effectiveness shown by improvements in timely reporting, and reductions in lead time and stock outs in the EM group can be also be attributed to the DPAT component, which laid emphasis on the use of data for decision making to drive continuous improvements and recognize good performance. DPATs facilitated much of the continued use of data by HF staff in EM districts for monitoring and management, and had a clear and strong association with improvements in all measures of effectiveness–– complete reporting rates were on average 20% higher in the EM group, lead times/responsiveness to orders were half as long and mean stock out rates half as high in EM compared to EPT districts. Compared to the paper based reporting system assessed at baseline, where districts only had access to HSA logistics data from 29% of HFs, now they are able to have timely access to data from over 80% of HSAs. Equally important, due to the design and acceptability of DPATs, HF and district managers use cStock data for resupply, for monitoring priority indicators such as reporting rates, and for management decision making to improve outcomes such as supply reliability. These findings show that not only is a feasible, acceptable system like cStock for supplying data beneficial, the higher performance on SC indicators in the EM group clearly points to the added value of DPATs, which, when integrated within existing health organizational systems and structures, can provide a mechanism for making data more meaningful and useful.Although there is a growing evidence base for implementation of mHealth systems to solve public health problems, only a limited number of solutions aim to improve supply chain effectiveness . To the The importance of exploring ways to promote routine system use and enhance acceptability is demonstrated by the SMS for Life study in Kenya and Tanzania, where financial incentives were provided for reporting stock information, resulting in higher reporting rates than in the Uganda study, where incentives were not used. The Uganda study however recognizes that better impact related to stock management was likely if district teams were fully engaged, emphasizing the importance of such reporting systems being nested within organization support structures. Similarly in Malawi, the implementation of cStock was integrated with additional components in the two intervention groups designed to enhance the overall performance of the supply chain; the findings from the EM group suggest that the team approach had an additive and significant effect in further improving SC performance for reporting rates, reporting completeness, lead times (responsiveness of the supply chain) and stock out rates (supply reliability). This finding is in agreement with the broader discussion by experts in the field of mHealth who have suggested that integration of mobile–based tools with existing health management systems and structures will be critical to national level adoption and scaling up of such programs . We beliOne of the greatest challenges in implementation and evaluation of cStock was related to the complex supply chain and product management environment in Malawi. Due to the economic crisis in the country iCCM implementing partners were provided funding by their donors to supplement the national supply of iCCM medicines and distribute products directly to HSAs. These parallel donor–funded distribution systems bypassed the government–funded public supply chain and therefore masked true product availability throughout the supply chain. Thus attribution of improvements in product availability as a result of the EM and EPT interventions was not possible to determine and supply reliability was used as an indicator instead. Additionally, the scope of this research was limited to analysis of effectiveness of cStock and DPATs (EM) in terms of supply chain outcomes, and did not delve into the additional cost and cost saving related to either component of this integrated approach.Our findings suggest that cStock is a feasible, acceptable and effective tool for improving community health supply chain management. The integration with DPATs to promote the timely and appropriate use of system generated data to strengthen community health supply chain outcomes should be considered as a context–specific adaption for countries interested in adopting cStock. We hypothesize that establishing multilevel teams serves to connect HSAs with decision makers at higher levels of the health system, align objectives, clarify roles in the supply chain and promote trust and collaboration, thereby laying the foundation for country ownership and scalability of a cStock–like system. Continued research around the factors contributing to scalability and sustainability, as well as cost–effectiveness of such interventions, will be critical to strengthen the evidence base in this area.


Ebola virus disease (EVD) outbreak in Nigeria has raised the level of awareness of both health care workers (HCWs) and members of community (MCs) on the threat posed by infectious diseases and need for improvement on infection control practices, but awareness of dangers of increasing antimicrobial resistance (AMR) remained low.To compare awareness level of HCWs and MCs on dangers of EVD and AMR and their control and to give educational intervention on dangers of AMR and its control to MCs with no prior knowledge of AMR.A cross-sectional survey of 195 HCWs and 265 MCs was conducted through structured questionnaire and interview.Majority of HCWs (95.4%) and MCs (82.8%) have recent knowledge of EVD dangers and give reasons like EVD way of killing, stigmatization, no drugs and vaccines as reason for their awareness. Only 17.2% of MCs were aware of AMR as problem, and only 3.4% of MCs and 10.3% of HCWs agreed that AMR is more deadly than EVD. However, 76.4% Drs, 95.1% nurses, 67.9% lab scientist, 66.7% pharmacists, 77.4% students and 100% of civil servants, drivers and religious leaders believed that EVD is more horrific and spread faster, while in reality its dangers is a drop in the ocean when compared with AMR. They both attributed the rapid awareness of EVD in Nigeria despite being new, to seriousness with which stakeholders and media fight EVD, the gesture AMR is yet to receive. All agreed that prevention, not treatment is the best option to tackle EVD. About 84.1% of HCWs and only 17.2% MCs believed that careful use of antibiotics can reduce cases of AMR. After short briefing of some MCs on dangers of AMR and its control through prudent use of antibiotics, a shift in belief from 17.2% to 48.7% was observed, with 64.8% civil servants, 54.1% students, 18.2% drivers 25.5% villagers and 25.0% traditional healers changed their method of control option to prevention rather than treatment.Despite extreme panic over EVD, awareness on dangers of AMR and its control remained very low among MCs. Efforts put in place during EVD outbreak by all stakeholders and the media need to be double to increase the knowledge of both HCWs and MCs toward AMR.None declared.


Although the vast majority respond positively to treatment, a significant proportion of PD patients develop daily fluctuations in mobility and troublesome involuntary movements known as levodopa-induced dyskinesias (LIDs). The time-to-onset and severity of this motor complication show large individual variability thus limiting the long-term use of levodopa and clinical strategies aimed at reducing LIDs manifestation. In the last few years, a considerable effort has been made to understand the neurobiological basis of this motor complication. In particular, recent evidence coming from human and animal studies has strongly contributed to reduce the gaps in our knowledge of LIDs pathogenesis.After its first use in clinic since 1960, oral administration of The papers in this research topic highlight several themes relevant to the understanding of the clinical and neurobiological basis of LIDs. From a neuropsychological perspective, Pietracupa et al.  have expFinlay et al.  systematBeyond the multiple phenomenology of LIDs in PD patients, further insights on the pathophysiology of hyperkinetic movement disorders derive from studies conducted in patients with idiopathic dystonia, tardive dyskinesia (TD), and Gilles de la Tourette syndrome (GTS). Dystonia is a hyperkinetic movement disorder characterized by sustained or intermittent muscle contractions causing abnormal, often repetitive, movements, postures, or both . The terInvolvement of cortical areas  as well of the cerebellum in patients with LIDs has been also suggested by transcranial magnetic stimulation (TMS) studies, specifically using protocols to probe synaptic plasticity in humans , 16, 17.The role of cortical areas such the IFC and the primary motor cortex and well of the cerebellum in the generation of LIDs is a novel concept supported by several recent studies reviewed in this research topic. Nevertheless, the cortico-striato-pallido-thalamo-cortical loop represents a critical network for the generation of LIDs as supported by the long-term beneficial effect of ablative surgery and deep brain stimulation (DBS) of the globus pallidus internus (GPi) and the subthalamic nucleus (STN) . AccordiThe current research topic is also enriched by three intriguing reviews related to the experimental models of LIDs. Animal models provide the unique opportunity to investigate in depth the molecular and cellular machinery involved in the pathophysiology of LIDs. Cenci  providesWith a different perspective, Morin and Di Paolo report relevant studies investigating glutamate receptor subtypes in relation to motor complications in PD patients and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned monkeys . MPTP-leIn conclusion, the contributions included in this research topic highlight the role of different cortical and subcortical areas as well as of other neurotransmitters beyond dopamine in the pathophysiology of LIDs. We hope that this research will stimulate new thinking about neurobiological mechanisms of LIDs.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Molecular Cell 57(1): 83–94, ISSN 1097-2765,


Coreopsis grandiflora (cgAUS1), catalyzing conversion of butein to sulfuretin in a type-3 copper center, is a rare example of a polyphenol oxidase involved in anabolism. Site-directed mutagenesis around the CuA site of AUS1 was performed, and recombinant enzymes were analyzed by mass spectrometry. Replacement of the coordinating CuA histidines with alanine resulted in the presence of a single copper and loss of diphenolase activity. The thioether bridge-building cysteine and a phenylalanine over the CuA site, exchanged to alanine, have no influence on copper content but appear to play an important role in substrate binding.Aurone synthase from  Laccases are multicopper oxidase enzymes, which catalyze the oxidation of phenolic and non-phenolic compounds by withdrawing the electron from the substrate and converting them in free radicals, which then polymerize Q9FRX6) was found in the 4-hydroxyaurone pathway of Antirrhinum majus  and Helichrysum bracteatum (Asteraceae) and catalyzes two reactions, the hydroxylation and the oxidative cyclization of 2′,3,4,4′,6′-pentahydroxychalcone (PHC) and 2′,4,4′,6′-tetrahydroxychalcone (THC) into aureusidin and bracteatin, respectively Coreopsis grandiflora (cgAUS1) is involved in the 4-deoxyaurone pathway and catalyzes only the conversion of chalcones to aurones, by oxidizing butein  to sulfuretin (4-deoxyaurone) Type-3 copper enzymes contain two copper atoms (CuA and CuB) and bind dioxygen in a characteristic “side-on” bridging mode (μ-ηcgAUS1) contains 602 amino acids, corresponding to a mass of 68 kDa cgAUS1). Typically the transit peptide of plant PPOs is cleaved in vivo after transport to the target location (in case of AUS1 as in most plant PPOs the lumen of the thylakoid membranes in chloroplast), thus the enzyme is expressed in the latent form cgAUS1 the putative cleavage region is near the SKE motif, marked in red in cgAUS1, see cgAUS1, shown in 93CAYC97 motif. This thioether-bridge was also found in both catechol oxidases of Ipomoea batatas (ibCO)  is present in all plant PPOs (catechol oxidases and tyrosinase). The bulky, monophenolase-blocking phenylalanine (F273 in cgAUS1) is present in most plant PPOs, but in some species leucine is at this position instead of phenylalanine, such as pineapple PPO from Ananas comosus and bread wheat PPO from Triticum aestivumAgaricus bisporus or Neurospora crassa and the fungal catechol oxidase from Aspergillus oryzae, but a thioether-forming cysteine is found in fungal tyrosinases at a different sequence location than in plant PPOs 16-19H in plant PPOs, see The total sequence (including the transit peptide) of aurone synthase (bCO) see  [15]. GeC.grandiflora (cgAUS1) was successfully heterologously expressed in Escherichia coli, but recombinant aureusidin synthase (AmAS1) has not been described so far Taraxacum officinale  heterologously expressed in E. coli and identified, through molecular modeling, a surface-exposed cysteine (C197 in the pro-enzyme see cgAUS1). Site-directed mutagenesis of this cystein to a serine proved this amino acid residue to stabilize this tetramer via a disulfide linkage aoTYR) A.oryzaebmTYR) of Bacillus megaterium were performed by Goldfeder et al. cgAUS1), expecting to have a decreased monophenolase activity. However, the monophenolase activity increased surprisingly Mutagenesis studies of recombinant PPOs are even more rare. In plant PPOs, the first site-directed mutagenesis study was performed of dandelion PPO-6 from cgAUS1 was previously described and the pro-enzyme was cloned in E. coli, thereby producing large amounts of pure, recombinant cgAUS1 cgAUS1 was isolated and purified from natural source as well and characterized and compared with the recombinant enzyme cgAUS1 cgAUS1 and therefore influences the monophenolase/diphenolase ratio ibCO revealed that the formed covalent bond increases structural restraints on the ligand sphere of the CuA center, therefore this cysteine (C97) was mutated to alanine in cgAUS1 C.grandiflora (cgAUS1) is presented in this work. In order to investigate the activity of all coordinating histidines, the three histidines of CuB were mutated to alanine.The full-length sequence of 22.1aurone synthase (cgAUS1) gene from C. grandiflora into pTrcHis2 vector was previously described Isolation and cloning of 2.2cgAUS1 enzymes was performed by inoculating with overnight culture, growing at 37 °C in SB culture medium , inducing with isopropyl-β-d-thiogalactopyranosid (IPTG) at an OD600 of 0.6 and harvesting the cells at an OD600 of ∼1.5, as described previously, g for 10 min and resuspended again in 10 ml 30 mM Tris–HCl, pH 8.5. The cells were lysed by three freeze–thaw cycles and by adding 0.3 mg/ml lysozyme  and 1 mM phenylmethanesulfonylfluoride (PMSF). After adding 0.05 mg/ml DNaseI (SigmaAldrich) and 10 mM MgCl2 the lysate was centrifuged  and directly loaded onto GSTrap FF.Production of wild type and mutated 2.3cgAUS1 pro-enzymes (wild type and mutants) were purified by applying the lysate to 5 ml GSTrap FF column . All proteins were loaded onto the column using 30 mM Tris, 150 mM NaCl at pH 8.0 as binding buffer. cgAUS1 was eluted from the column using the same buffer supplemented with 10 mM reduced glutathione. The eluted peak was collected and buffer exchange to the binding buffer, to remove the glutathione, was performed with Vivaspin 20 30.000 MWCO PES ) . The GST-tag was cleaved by PreScission Protease  at 4 °C over night. The protein solution containing the GST-tag and the untagged, pure protein was loaded again onto the GSTrap FF column. GST-tag was bound onto the column and the untagged protein was eluted. The pure, untagged protein was concentrated with Vivaspin 20 30.000 MWCO PES and stored at 4 °C in 10 mM sodium acetate (pH 5.0) for further use.The GST-tagged 2.4cgAUS1 enzymes was measured after cell lysis and monitored by spectrophotometric measurements (SHIMADZU UV-1800). Monophenolase activity was determined by the use of 1 ml of 10 mM sodium phosphate buffer, pH 6.5, 2.5 mM SDS as an activating agent, 33 μM l-tyrosine as substrate and up to 5 μL of enzyme solution  were mixed with reduced loading buffer and heated for 10 min before loading to the gel.Electrophoresis was performed by the method of Laemmli In-gel AUS activity was determined by applying partially 8% SDS–PAGE according to Cabanes et al. 2.62O, 2% acetonitrile, 0.1% formic acid).2 μg of purified protein were applied to SDS–PAGE and visualized by coomassie staining followed by excision of gel bands corresponding to the protein. After band excision destaining, washing, reduction and carbamidomethylation of cysteines was performed. In-gel digestion was achieved using chymotrypsin at 37 °C over night. Peptides were recovered from the gel using ultrasonication and dried completely by vacuum centrifugation. The samples were stored at −20 °C prior to LC–MS/MS analysis. Dried peptide samples were dissolved in 5 μL 30% formic acid (Fluka) and diluted with 40 μL eluent A . Data analysis was performed via Proteome Discoverer 1.4 by searching against the corresponding mutant sequences using Sequest as search engine. Peptide mass tolerance was 5 ppm and the fragment mass tolerance 0.5 Da. Modifications applied for each search were carbamidomethylation as fixed modification for cysteines and methionine oxidation as variable modification. For high confidence of the MS data, the false discovery rate (FDR) of the peptide spectrum matches (PSM) was set to <0.01 (Proteome Discoverer). Details about applied parameters can be found in 2.7cgAUS1 wild type and mutants  and digested for one hour at 100 °C in the oven. After cooling to room temperature the loss of liquid was filled up with deionized water. Samples were then measured directly with F-AAS . Samples containing less than 0.2 mg/L Cu were measured by means of GF-AAS  using a Pd-Mg modifier. For both methods, element concentrations of each sample were calculated from the corresponding regression lines (correlation factor > 0.9995) using five different dilutions of a standard solution . The range of calibration was 0.2–5.0 mg/L for F-AAS and 1.00–50.0 μg/L for GF-AAS, respectively.Pure  mutants  were use mutants  were aci33.1cgAUS1 proenzyme into pGEX-6P-1 the recently developed method could be advanced to successfully produce large amounts of pure, active and soluble cgAUS1 wild type (58 kDa + 26 kDa GST tag) in E. coli and simplify the purification process by a reduction of the four ion exchange purification steps to only one affinity chromatography step cgAUS1 wild type is shown in cgAUS1 was eluted by the use of reduced glutathione. All cgAUS1 mutants  were expressed and purified under identical conditions, using wild type cgAUS1 as control sample. The amount of soluble, expressed and purified enzyme, shown in cgAUS1. The C97A mutant showed almost a less than one-tenth ratio (0.06 mg/ml) followed by F273A (0.27 mg/ml), H286A (0.33 mg/ml), H93A (0.37 mg/ml), H125A (0.74 mg/ml), H116A (1.09 mg/ml), H256A (1.20 mg/ml) and H252A (1.24 mg/ml) compared to cgAUS1 wild type (0.63 mg/ml). The significantly lower concentration of C97A may be due to the missing thioether bond, between C97 and H116. In contrast, Gerdemann et al. As a result of recloning 3.2cgAUS1 samples were used to determine the incorporated copper content by AAS  contained only 1 Cu per molecule enzyme. For the F273A mutant, 2 Cu per molecule cgAUS1 could be determined, as it is the case in the wild type. The C97A mutant contained as well two Cu per molecule cgAUS1.The purified t by AAS . The copaoTYR the substitution of cysteine (C82) with alanine as well as substitution of the three copper coordinating histidines  in the CuA site decreased copper binding to 50%, indicating that these mutants contain only 1 Cu per molecule cgAUS1, where mutation of the cysteine residue did not decrease the copper content, indicating that the thioether bond seems to be structurally important for expression and folding, as well as for enzymatic catalysis  from A.bisporusabPPO4 at the position of the phenylalanine in ibCO, which endorsed the assumption of a blocker residue cgAUS1 disproves this role for the phenylalanine, as blocking residue for monophenolated substrates. This indicates, that phenylalanine 273 plays a role in substrate binding. Investigation on a less bulky valine, of bmTYR, at the same position of the phenylalanine showed an increase of the monophenolase activity after mutation of this valine to a phenylalanine bmTYR mutant does as well contradict a previously suggested proposal of phenylalanine as a general blocking residue in PPOs, which would eliminate the ability of the PPO to perform hydroxylation of monophenols The bulky CuA-site blocking phenylalanine residue (F273) was assumed to hinder monophenolated substrates in catechol oxidases aoTYR site-directed mutagenesis of all copper coordinating histidines to asparagine, a single-essential cysteine residue to alanine  and the non-coordinating histidine in CuB as well to asparagine showed no detectable activity after mutation cgAUS1. Furthermore, both single mutations (the C97A and the H116A) demonstrated in cgAUS1 that this thioether bond is essential for the diphenolase activity In 4E. coli. We propose in vivo a twofold importance for C97: for proper folding of the protein structure and for diphenolase activity. The decrease of the copper content in the histidine mutants  was expected, but surprisingly the loss of one copper coordinating histidine had no influence on the expression as soluble protein. However, we demonstrate that ligation of the copper at CuA site is only mediated by the three histidine residues , but is not influenced by the thioether bond. The assumption that the F273 residue acts as a blocker residue for substrates or hinders the monophenolase activity could be disproved for cgAUS1, as it still contains two copper atoms per molecule but lost its total activity cgAUS1 that the amino acid F273 is essential for substrate binding, but is not able to change the reaction mechanism to a tyrosinase-like enzyme.A summary of the presented results is shown in The authors declare no competing financial interest.


In this study, TiO2-vermiculite composite nanofibers with a mesh structure and a diameter of approximately 300 nm were prepared via sol–gel approach combined with electrospinning technique. The samples were characterized by X-ray diffraction, scanning electron microscopy, ultraviolet–visible spectroscopy, etc. The photocatalytic property was also evaluated. The TiO2-vermiculite composite nanofibers annealed at 550 °C for 3 h exhibited the best absorption and photo-degradation ability for the treatment of methylene blue. The results implied that the combination of mineral vermiculite powders with TiO2 enhanced the absorption-degradation performance of the as-prepared photocatalytic materials, consequently promoting the materials’ ability to degrade methylene blue.Titanium dioxide (TiOThe online version of this article (doi:10.1186/s11671-015-0977-1) contains supplementary material, which is available to authorized users. Several methods have been reported to synthesize TiO2 nanoparticles, nanowires, nanotubes, nanofibers, etc. However, most of the photocatalytic materials currently used consist of nanoparticles [2 is 3.2 eV, which is an intrinsic property of the material. Decreasing the energy band would allow for the activity of TiO2 by UV light, which accounts for 5 % of the whole spectrum of sunlight. Therefore, broadening the responding spectral region of TiO2 is an effective method to increase the photocatalytic property of such materials [Titanium dioxide  under irradiation with UV light. The results showed that the utilization of TiO2 composite nanofibers containing 2 wt.% of vermiculite resulted in a remarkable absorption and an enhanced degradation of MB.In this study, vermiculite and TiO2-vermiculite composite nanofibers was the preparation of a precursor via the sol–gel process. To fabricate the precursor, 1.5 g of polyvinyl pyrrolidone , 20 ml of ethanol, 5 ml of acetic acid, and 5 ml of tetrabutyltitanate were mixed with different amounts of vermiculite powders and stirred for 2 h to ensure a homogeneous mixture. The obtained precursors were then transferred into the electrospinning instrument to fabricate the vermiculite/PVP/Ti(OCH(CH3)2)4 nanofibers. An electrical potential of 15 kV was applied between the nozzle and the collector in order to eject the sol–gel precursor to the collection board which was installed a distance of 15 cm to the nozzle. In order to obtain nanofibers with a homogenous size distribution, the propulsion speed was set to 2 ml/h. After a thermal treatment for 3 h at 550 °C, the vermiculite/PVP/Ti(OCH(CH3)2)4 fibers were transformed into TiO2-vermiculite composite nanofibers.The initial step in the synthesis of the TiO3)2)4 fibers were characterized by the simultaneous application of thermogravimetry and differential scanning calorimetry (TG-DSC). The phase composition of the TiO2-vermiculite composite nanofibers was studied by D/max-rA X-ray diffraction . The microstructures and nanostructures of the nanofibers were characterized by scanning electron microscopy  and transmission electron microscopy . The nanofibers were ultrasonically dispersed in ethanol and then dropped onto carbon-coated copper grids prior to the TEM investigations. Diffuse reflectance ultraviolet–visible (UV–vis) absorption spectra were recorded using a Carry 5000 UV–vis-NIR spectrophotometer with an integrating sphere attachment.The vermiculite/PVP/Ti and the solution was stirred for 30 min under exclusion of light. The adsorption equilibrium of the solution was reached when the methylene blue concentration remained in balance. Then, the solution was stirred by magnetic stirring and irradiated with UV light using a 500 W high-pressure mercury lamp performance in order to evaluate the photocatalytic property. A 3 ml of the pollutant solution was sampled every 20 min and centrifuged for the separation of the upper clear solution. The concentration of MB was analyzed using a UV–vis spectrophotometer  at the characteristic wavelength of 664 nm.3)2)4 composite nanofibers are shown in Fig. 3)2)4. The formation of the anatase phase of TiO2 began at a temperature of 475 °C. The phase transformation from the anatase phase to the rutile phase was completed for temperatures above 525 °C [The results of TG-DSC analyses of the PVP/Ti, the TiO2-vermiculite composite nanofibers show an optimized photocatalytic property.Both the anatase and the rutile phase were found in the composite nanofibers exposed to the thermal treatment at 550 °C Fig. c. In addproperty , 13. The2-vermiculite composite nanofibers for the degradation of MBin a solution with an MB concentration of 10 mg/L. As shown in Fig. 2 nanofibers, for the TiO2 composite nanofibers containing 2 wt.% vermiculite, the reaction equilibrium is reached earlier.Figure 2 nanofibers with 2 wt.% vermiculite nanoparticles is the highest among all samples. This can be explained as follows: The degradation process is caused by the TiO2. With an increasing amount of vermiculite and a decreasing amount of the catalyst (TiO2), the absorbability is enhanced, whereas the catalytic ability is reduced at the same time. Therefore, the TiO2 nanofibers with 2 wt.% vermiculite nanoparticles show stronger capabilities  to treat chemical pollutions.The photodegradation rate is an important factor for the evaluation of photocatalytic materials. Figure 2 composite nanofibers with different vermiculite mass fraction are compared in Fig. Eg) for each sample was obtained by plugging the absorption data into the direct transition equation:α - optical absorption coefficient;υ - photon energy;Eg - direct band gap;h and ED - a constant.The UV–vis absorption spectra of the TiOEg) of the samples, which are 3.05, 3.14, 3.08, 3.17, and 3.18 eV for a vermiculite mass fraction of 0, 2, 4, 10, and 50 wt.%, respectively [Additional files 2 nanofibers is 3.05 eV due to the band alignment of the rutile and anatase TiO2 phase. Moreover, the addition of a small amount of vermiculite to the TiO2 nanofibers significantly changed the band gap energy. However, adding a larger amount of vermiculite to the nanofibers had negative effect on the band gap. In this paper, the composite nanofibers with 2 wt.% vermiculite showed the most suitable direct band gap and the highest photocatalytic activity.The tangent to the curves Fig. b at the 2-vermiculite nanofiber composites were synthesized by combining a sol–gel process with the electrospinning technique. The TiO2-vermiculite nanofiber composites were obtained after thermal treatment at 550 °C for 3 h. The results of the phase composition analysis indicate that the main phase of the prepared samples was the anatase phase, and a small amount of the rutile phase could also be detected. The analysis of the structure of the composite nanofibers revealed a smooth surface and diameter of approximately 300 nm. The photodegradation of methylene blue by the as-prepared TiO2-vermiculite composite nanofibers was performed under irradiation lights. In this study, the addition of the mineral powder to the TiO2 nanofibers was demonstrated to enhance the adsorption-photocatalytic performance of the photocatalytic material.TiO


This study evaluated the potential effect of trastuzumab on the electrocardiogram (ECG) QT interval and assessed the potential pharmacokinetic interaction between trastuzumab and carboplatin. Here, we report the QT and safety results.Patients with metastatic or inoperable HER2-positive solid tumors received docetaxel and carboplatin on Day 1 of each 3-week (q3w) cycle. Trastuzumab was administered intravenously, as an accelerated loading dose regimen, on Cycle 1, Day 2 and Cycle 1, Day 8, and then on Day 1 of each subsequent q3w cycle. ECG assessments were performed pre- and posttrastuzumab infusion in the first two cycles. Fridericia’s correction was applied to QT intervals (QTcF). Baseline-adjusted QTcF intervals (the change from baseline) and their 90 % confidence intervals (CIs) were calculated.The study enrolled 59 patients. At all time points, the 90 % CI upper bound for the mean baseline-adjusted QTcF was <10 ms. At steady-state serum trastuzumab concentrations, the mean baseline-adjusted QTcF interval was −8.4 ms . No patient exhibited an absolute QTcF interval of >480 ms. No relationship was observed between trastuzumab concentration and baseline-adjusted QTcF interval. At data cutoff, 84.5 % of patients had experienced grade ≥3 adverse events, the most common of which were hematologic and as expected. Left ventricular ejection fraction remained ≥45 % in all patients during the study.The results suggest that trastuzumab had no clinically relevant effect on QTcF interval. The safety profile of trastuzumab in combination with carboplatin and docetaxel was consistent with the known safety profile of this combination.The online version of this article (doi:10.1007/s00280-014-2603-9) contains supplementary material, which is available to authorized users. Hoffmann-La Roche, Basel, Switzerland) binds to HER2, inhibiting ligand-independent HER2 signaling and promoting antibody-dependent cell-mediated cytotoxicity of HER2-positive cancer cells. Trastuzumab improves survival outcomes when added to chemotherapy for the treatment of patients with HER2-positive early breast cancer (EBC)  related to prior systemic therapy, surgery, or radiotherapy, peripheral sensory neuropathy with functional impairment (NCI-CTCAE grade >2) due to factors other than prior anticancer treatment, a life expectancy <12 weeks, trastuzumab therapy within 100 days, or any investigational agent within 28 days prior to the start of study treatment.2 and target area under the concentration–time curve (AUC) of 6 mg/ml/min , ondansetron hydrochloride [14 (26.4 %)], and prochlorperazine [12 (22.6 %)].QT interval duration was negatively correlated with heart rate and positively correlated with RR interval duration, as shown in Supplementary Fig. S1. QT intervals were corrected using Fridericia’s (QTcF) and Bazett’s (QTcB) methods. Using Fridericia’s correction, the effects of heart rate and RR interval on QT interval were reduced, as shown by plotting individual average QTcF intervals against time-matched heart rates and RR intervals . This produced linear-fitted lines with small gradients, thus demonstrating no apparent relationship between QTcF interval and heart rate or RR interval, and thereby confirming the suitability of the QTcF correction method. In contrast, more variability was observed when using Bazett’s correction, with QTcB intervals overcorrected at higher heart rates and undercorrected at lower heart rates .The mean baseline-adjusted  average QTcF interval (90 % two-sided CI) at each ECG assessment time point is shown in Fig. At steady-state trastuzumab concentrations , the mean baseline-adjusted QTcF interval was −8.4 ms . Similarly, the mean baseline-adjusted QTcB interval at trastuzumab steady-state was −5.9 ms .The proportions of patients within absolute QTc interval categories, as defined by ICH E14 guidance, at each time point are presented in Table The mean baseline-adjusted average PR interval increased over time to a maximum of 13.7 ms at the Cycle 2, Day 1, 30 min postinfusion time point. Overall, however, baseline-adjusted secondary ECG parameters showed no obvious trends that would suggest a clinical risk of cardiac dysrhythmia  trastuzumab concentration was 0.2  μg/ml; the mean Cycle 1, Day 8 and Cycle 2, Day 1 postinfusion concentrations were 186.4  and 179.1  μg/ml, respectively. The concentration–QTc dataset consisted of 297 ECG assessments with parallel trastuzumab concentration measurements from 53 patients, and contained data collected at trough and peak steady-state trastuzumab concentrations. A linear least-squares fit of trastuzumab concentration versus baseline-adjusted QTcF interval produced a slope of linear fit with a small gradient, close to 0 (0.0009), indicating no apparent relationship between trastuzumab concentration and baseline-adjusted QTcF interval  was 4.4 months (range 0.0–37.7 months). Patients had received a median of seven (range 1–52), four (range 1–24), and four (range 1–51) doses of trastuzumab, carboplatin, and docetaxel, respectively, and the median duration of trastuzumab treatment was 15 weeks (range 0–163 weeks). Fifty-seven out of 58 safety-evaluable patients had received at least one dose of trastuzumab.A total of 57 out of 58 safety-evaluable patients 98.3 %) experienced AEs of any grade, and 49 patients (84.5 %) had experienced grade ≥3 AEs, the most common of which (in ≥4 patients) are shown in Table  % experiEight patients experienced cardiac AEs. The mean LVEF at baseline was 64.0 % [standard deviation (SD): 8.6 %], and the mean lowest LVEF value postbaseline was 59.5 % (SD: 6.6 %). Forty-four patients (93.6 %) had LVEF values of ≥50 % at all postbaseline time points; no postbaseline values of <45 % were observed during the study. Two patients experienced grade 2 left ventricular dysfunction. One of these events, considered by the investigator to be related to trastuzumab, resolved after 21 days and had no associated ECG changes. The other left ventricular dysfunction event, considered to be unrelated to trastuzumab, was ongoing at the end of the study. The patient had a history of left bundle branch block and had experienced an AE of prolonged QT interval several weeks prior to the left ventricular dysfunction event. This patient’s prolonged QT interval was an isolated grade 1 AE. Prior to this event, the patient experienced grade 2 hypocalcemia but did not receive the protocol-required calcium supplementation (and was therefore a protocol violator).The results of this study suggest that trastuzumab had no clinically relevant effect on the duration of the QTcF or QTcB intervals in patients with HER2-positive metastatic or locally advanced, inoperable solid tumors. At the first postinfusion ECG assessment, the mean QTcF interval was increased slightly from baseline; at all subsequent time points, mean QTcF intervals were reduced from baseline. No patients had absolute QTcF interval durations that were within the range of clinical concern, i.e., there were no absolute values of >480 ms or increases from baseline of >30 ms. Additional ECG parameters  were not altered. Importantly, sustained QT interval prolongation did not occur even though a substantial proportion of patients received at least one restricted concomitant medication with a known risk of prolonging the QT interval. Furthermore, there was no relationship observed between serum trastuzumab concentration and change from baseline in QTcF interval duration. It should be noted that this study employed an unapproved (off-label), accelerated trastuzumab loading dose regimen to achieve steady-state serum trastuzumab concentrations early, in order to test more rigorously for potential effects on QT interval.Increased rates of cardiac dysfunction were an unexpected observation during the clinical development of trastuzumab, influencing the design of subsequent trials, in which LVEF was monitored more frequently and stricter cardiac eligibility criteria were applied , 21, 22.®, F. Hoffmann-La Roche, Basel, Switzerland) and pertuzumab , have also demonstrated no effect on QT interval [Previous reports evaluating QT interval during trastuzumab-based therapy have been conflicting. An observational study reported long-term treatment with trastuzumab to be associated with significantly prolonged QT interval. However, this study compared patients with HER2-positive and HER2-negative EBC. Furthermore, the results were confounded by the use of anthracycline-based chemotherapy, which is known to affect cardiac function . In anotinterval , 30. Theinterval . Small mEvaluation of the potential effect of trastuzumab on QT interval using a thorough QT study, as defined by ICH E14 guidance , was notIn this study, trastuzumab in combination with carboplatin and docetaxel had an acceptable safety profile which was comparable to the known safety profile of the trastuzumab, carboplatin, and docetaxel regimen in patients with HER2-positive EBC . No QT pSupplementary material 1 (DOCX 13 kb)Supplementary Fig. S1 Uncorrected QT, QTcF, and QTcB intervals by heart rate and by RR interval. QTcB, QT interval corrected using Bazett’s correction; QTcF, QT interval corrected using Fridericia’s correction. (A) Uncorrected QT interval plotted against heart rate with line of linear fit ; (B) Uncorrected QT interval plotted against RR interval with line of linear fit ; (C) QTcF interval plotted against heart rate with line of linear fit ; (D) QTcF interval plotted against RR interval with line of linear fit ; (E) QTcB interval plotted against heart rate with line of linear fit ; (F) QTcB interval plotted against RR interval with line of linear fit . (EPS 3069 kb)Supplementary Fig. S2 Box plots of baseline-adjusted QTcF interval by nominal electrocardiogram time point. QTcF, QT interval corrected using Fridericia’s correction. The standard span is 1.5× (interquartile range). Whiskers are drawn to the nearest value not beyond a standard span from the quartiles. Points beyond this (outliers) are drawn individually (EPS 811 kb)Below is the link to the electronic supplementary material.


The structural, morphological and ammonia sensing properties of the prepared composite nanofibers were studied using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), four-point probe techniques, X-ray diffraction (XRD) and a home-made gas sensing test system. All the results indicated that the CSA concentration had a great influence on the sensing properties of CSA-doped PA6/PANI composite nanofibers. The composite nanofibers doped with 0.02 M CSA showed the best ammonia sensing properties, with a significant sensitivity toward ammonia (NH3) at room temperature, superior to that of the composite nanofibers doped with 0.04–0.10 mol/L CSA. It was found that for high concentrations of CSA, the number of PANI–H+ reacted with NH3 would not make up a high proportion of all PANI–H+ within certain limits. As a result, within a certain range even though higher CSA-doped PA6/PANI nanofibers had better conductivity, their ammonia sensing performance would degrade.Camphor sulfonic acid (CSA)-doped polyamide 6/polyaniline (PA6/PANI) composite nanofibers were fabricated using  It was found that PANI CE polymerized with 0.35 M H2SO4 shows the best photovoltaic performance with a solar-to-energy conversion efficiency of up to 5.57%. Arenas et al. [et al. [et al. [in situ polymerization of aniline with CSA as dopant, and then the sensors based on micro/nano-PANI were directly fabricated onto the interdigital electrodes by a self-assembly method. Zhou et al. [m-cresol, and then the product was cast on slides to obtain CSA-PANI films. However, many of the materials mentioned above are powders, and for many functional materials, especially for those applied to sensors, specific surface area is very important. Nanofibers have larger specific surface area and their shapes are controllable. There are already several published papers dealing with the preparation of PANI-based nanofibers. Nevertheless, there are few reporting the effects of CSA concentration on the ammonia sensing properties of PANI-based composite nanofibers.Gas sensors are important in environmental monitoring, home safety and chemical control . Conductet al.  fabricats et al.  synthesi [et al.  prepared [et al.  synthesiu et al.  dissolvein situ polymerization of aniline under different CSA concentration  conditions with electrospun PA6 nanofibers as templates. The ammonia sensing properties of the composite nanofibers and the sensing mechanism were studied and discussed.In this work, for the first time, CSA-doped PA6/PANI composite nanofibers were prepared as ammonia sensors by 2.2.1.4 g/mol) was obtained from Zig Zheng Industrial Co. Ltd. . Formic acid (FA), aniline monomer, ammonium persulfate (APS) and ammonium hydroxide (NH3·H2O) were purchased from Sinopharm Chemical Reagent Co. Ltd. . (1S)-(+)-10-Camphorsulfonic acid (CSA) was purchased from Aladdin Reagent Database Inc. . All chemicals and reagents were used as received, except for aniline monomer, which was distilled twice under reduced pressure before use. Distilled water was used in this study.Polyamide 6 (PA6)  were washed with deionized water followed by acetone, and dried in vacuum at 50 °C for 24 h.2.3.−1 with a 4 cm−1 spectral resolution by using a NEXUS 470 spectrometer . The conductive properties of the composite nanofibers and composite nanopowders were investigated by four-point probe techniques. The crystalline structure analysis was also performed on a D8 Advance X-diffractometer , over the 2θ range of 3°–90°. The particle diameter was measured by Brookhaven Instruments .The surface morphologies of the CSA-doped PA6/PANI composite nanofibers were investigated using a field emission scanning electron microscope . Fourier transform infrared (FTIR) spectra were obtained in the range of 4000–400 cm2.4.3 were investigated with a home-made test system as shown in 2 were pasted onto the open area between the two electrodes. The tests were performed at room temperature (25 ± 1 °C). The actual ammonia volumes injected into the air chamber were 0.3367, 0.6734, 1.3468, 2.0202, 2.6936 and 3.3670 μL, corresponding to ammonia vapor with the concentration of 25, 50, 100, 150, 200 and 250 ppm respectively. The resistance of the composite nanofbiers was measured using an Agilent-34401A data acquisition system (Agilent Technologies (China) Co. Ltd., Beijing, China). The resistance of CSA-doped PA6/PANI composite nanofibers in air (R0) and in the presence of NH3 (Ri) were measured to evaluate the gas response, S, defined as S(%) = (Ri − R0)/R0 × 100.The sensing behaviors of the samples to NH3.3.1.For PANI, fiber structure would provide a big specific surface area which could help improve the sensitivity of a gas sensor . The sur3.2.−1 [−1 assigned to the stretching vibration of the C-C in the benzene ring and the peak around 1559 cm−1 originated from stretching vibrations of C=N and C=C bonds in quinone units can be found. These characteristic peaks also existed at the spectra of the CSA-PA6/PANI composite nanofibers. In addition, the stretching vibration peak at 1637 cm−1 assigned to the C=O stretching of PA6 can be found at the spectra of the CSA-PA6/PANI composite nanofibers, too. It can also be observed that the characteristic peaks of the prepared composite nanofibers shifted slightly. This may be the results of the differences in doping percentage.−1  can be f3.3.The X-ray diffraction patterns of CSA-PANI nanopowders with different doping degrees are presented in 3.4.+ captured by PANI chains. However, the sensitivity of the prepared nanofibers did not become higher when the nanofibers' conduction was improved.This showed that when the CSA concentration was in the range of 0.02–0.10 M, the higher the CSA concentration was, the better conduction the materials had, which was because there were more H3.5.3.5.1.3 are shown in 3. A moment later, the resistance became stable. When the gas room was opened and fresh air was introduced, the resistance decreased and returned to the original resistance gradually.The changes in resistance of CSA-doped PA6/PANI composite nanofibers with respect to time on the exposure of NH3 gas. The response of CSA-doped PA6/PANI composite nanofibers was enhanced with increasing concentration of NH3 gas from 25 to 250 ppm operating at room temperature. The CSA-doped PA6/PANI composite nanofibers gas sensors all showed a good sensitivity to ammonia vapor, and the gas sensor of 0.02 M CSA-doped PA6/PANI composite nanofibers possessed the highest response amplitude to ammonia among the five sensors tested. The response values are depicted in 3 concentration was 100 ppm, the response value of 0.02 M CSA-PA6/PANI was 253.96%. This was superior to the results  recently reported by Pang et al. [et al. [et al. [g et al. , Pan et  [et al. .3.5.2.3. The sensitivity of the composite nanofibers increased with increasing concentration of NH3 gas from 25 to 250 ppm and it was found to be 82.67%, 161.16%, 253.96%, 326.54%, 363.90%, 436.98%, respectively operating at room temperature. The repeatability is a very important property of gas sensor. The response of 0.02 M CSA-doped PA6/PANI composite nanofibers gas sensor was monitored for repeated exposure and removal of 250 ppm ammonia up to five cycles, as shown in Because the 0.02 M CSA-doped PA6/PANI composite nanofibers gas sensor showed the highest sensitivity among the samples tested, the other properties of 0.02 M CSA-doped PA6/PANI composite nanofibers were also examined. 3.It can be observed that the prepared CSA-doped PA6/PANI composite nanofibers sensor had high repeatability. In addition, to test the cross sensitivity, 0.02 M CSA-doped PA6/PANI composite nanofibers were exposed to 250 ppm acetone, ethanol and methanol, respectively, at room temperature. As shown in 3.5.3.The sensing mechanism of CSA-doped PA6/PANI composite nanofibers is illustrated in 3, PANI was de-doped and the reversible reaction [+ + NH3 ⇌ PANI + NH4+ ocurred. As a result, the resistance of PANI changed, embodied as sensitivity. To improve the conductivity of PANI, high concentrations of CSA were introduced, within certain limits. High concentrations of CSA would improve the conductivity of PANI and more PANI–H+ would exist. When the NH3 concentration was constant, the number of NH3 molecules was also constant under certain conditions. This resulted in a certain amount of the PANI–H+ which could react with NH3. Then, when the concentration of CSA was high, the PANI–H+ reacted with NH3 would not make up a high proportion of all PANI–H+ within certain limits. As a result, the resistance change of the tested sensor would not account for a big percentage of the initial resistance. That is, the value of S(%) = (Ri − R0)/R0 × 100 was not very obvious, which appeared as low sensitivity. In addition, when the large molecule protonic acid CSA modified the PA6/PANI composite nanofibers as a dopant, it would increase the steric hindrance of the de-doping reactions. Therefore, when the concentration of CSA increased, the response values of the composite nanofibers decreased.When exposed to NHreaction : PANI–H+4.3 gas at room temperature. The gas sensing properties of the composite nanofibers to NH3 indicated that these thin films of CSA-doped PA6/PANI composite nanofibers were good candidates for NH3 detection. It was also found that for high concentrations of CSA, the number of the PANI–H+ reacted with NH3 would not make up a high proportion of all PANI–H+ within certain limits and steric hindrance would reduce NH3's chances of being absorbed by PANI. As a result, even though high concentration CSA-doped PA6/PANI nanofibers had better conductivity, their ammonia sensing performance was degraded.The present work reported the preparation of different concentration CSA-doped PA6/PANI composite nanofibers for the detection of NH