To detect all malaria infections in elimination settings sensitive, high throughput and field deployable diagnostic tools are required. Loop-mediated isothermal amplification (LAMP) represents a possible field-applicable molecular diagnostic tool. However, current LAMP platforms are limited by their capacity for high throughput.Plasmodium falciparum parasites was used to determine the limit of detection of the HtLAMP assay, using P. falciparum (HtLAMP-Pf) and Plasmodium genus (HtLAMP-Pg) primers, on whole blood and filter paper, and using different DNA extraction protocols. The diagnostic accuracy of HtLAMP was validated using clinical samples from Papua New Guinea, Malaysia, Ghana and The Gambia and its field applicability was evaluated in Kota Marudu district hospital, Sabah, Malaysia.A high-throughput LAMP (HtLAMP) platform amplifying mitochondrial targets using a 96-well microtitre plate platform, processing 85 samples and 11 controls, using hydroxynaphtholblue as a colourimetric indicator was optimized for the detection of malaria parasites. Objective confirmation of visually detectable colour change results was made using a spectrophotometer. A dilution series of laboratory-cultured 3D7 The HtLAMP assay proved to be a simple method generating a visually-detectable blue and purple colour change that could be objectively confirmed in a spectrophotometer at a wavelength of 600 nm. When compared with PCR, overall HtLAMP-Pg had a sensitivity of 98 %  and specificity 83 % . HtLAMP-Pf had a sensitivity of 97 %  and specificity of 96 % . A validation study in a regional hospital laboratory demonstrated ease of performance and interpretation of the HtLAMP assay. HtLAMP-Pf performed in this field setting had a sensitivity of 100 %  and specificity of 95 %  compared with multiplex PCR. HtLAMP-Pf also performed well on filter paper samples from asymptomatic Ghanaian children with a sensitivity of 88 % .This colourimetric HtLAMP assay holds much promise as a field applicable molecular diagnostic tool for the purpose of malaria elimination.The online version of this article (doi:10.1186/s12936-015-0848-3) contains supplementary material, which is available to authorized users. Malaria elimination is an ambitious but enticing goal. Elimination is defined as “interrupting local mosquito-borne malaria transmission in a defined geographical area, i.e. zero incidence of locally contracted cases, although imported cases will continue to occur” . By defiP. falciparum and P. vivax, passive and active detection of infection in the field have relied on two diagnostic tools: thick and thin blood film microscopy, and immunochromatographic detection of circulating parasite antigens using rapid diagnostic tests (RDT). While expert microscopy has been shown to have 100 % sensitivity at thick film parasite densities of 50 parasites/µL, sensitivity decreases significantly under operational field conditions [Plasmodium species, compared to detection by PCR [For nditions . Furthern by PCR . It has n by PCR . The sucn by PCR . The tarn by PCR , a levelBacillus stearothermophilus (Bst) polymerase enzyme. It does not require the cyclical temperature changes [Loop-mediated isothermal amplification (LAMP) is a nucleic acid detection technology that has the potential to significantly improve malaria field diagnostics, particularly in elimination settings. LAMP differs from PCR in several critical ways. Firstly, it is an isothermal process relying on the  changes  necessar changes  or using changes , hydroxy changes  and pico changes . LAMP en changes , a biolu changes , a later changes  and a po changes .P. falciparum using crudely extracted DNA from whole blood [P. knowlesi [P. falciparum [P. vivax [P. falciparum [P. vivax using a combination of pan-genus and P. falciparum-specific LAMP primers [Since its first description in 2001  LAMP hasle blood , or usinle blood . Subsequle blood , includiknowlesi , 26, andlciparum , 27 and P. vivax , 29 can lciparum , includilciparum  and for  primers . However primers  for the  primers . The dem primers  suggestsThis paper describes development of a platform for malaria LAMP that increases the throughput of the assay, and simplifies DNA extraction and sample analysis, resulting in improved field applicability. Validation of the platform was carried out by investigating its diagnostic accuracy using PCR as a gold standard as it is highly sensitive and widely available in reference laboratories for detecting low-level parasitaemia.P. falciparum parasites was prepared in 50 % haematocrit blood using initial samples with parasitaemia adjusted to 1 and 0.5 %, as determined by microscopic examination of thin blood films. The number of parasites per microlitre was calculated using an estimate of 5 × 106 red cells/µL to create a dilution series of samples ranging from 5000 to 0.5 parasites/µL. Four 5 µL blood spots at each dilution were dried for 48 h on blotting paper (Whatman 3MM) before being stored at 4 °C with desiccant. Whole blood was stored at −20 °C.A tenfold dilution series of laboratory-cultured 3D7 For assay validation, clinical samples were retrospectively made available from the following clinical trials: (1) filter paper samples (n = 25) from asymptomatic children from Ghana , 34, (2)The study protocols for each study were approved by the Ghana Health Service Ethics Committee  and the Ethics Committee of the London School of Hygiene and Tropical Medicine , Joint Gambia Government/Medical Research Council Ethics Committee (re. 2002/910), QIMR Berghofer Medical Research Institute Human Research Ethics Committee (P2119), the Malaysian Medical Research Ethics Committee and Menzies School of Health Research, respectively.The chelex method for DNA extraction from filter paper  was furtFor whole blood, the chelex method  was furtThe Gambia whole blood samples were extracted using the WB-chelex protocol on 5 µL of blood. The filter paper samples from Ghana were extracted using the FP-chelex standard protocol. The PNG and Sabah filter paper samples were extracted using the FP-chelex rapid protocol. The differences in methods used for clinical sample processing reflect the different sample sets used to validate each of the extraction protocols.4SO4), 1.25 mM each dNTP, 3.55 µM each Pf869FIP/BIP, 1.78 µM each Pf869LF/LBn, 0.2 µM each Pf869F3/B3, 1.18 µM each PgID19FIP/BIP, 0.59 µM each PgID19 LF/LB and 0.15 µM each PgID19 F3/B3, 120 µM Hydroxynaphthol-blue  and 8 units Bst polymerase . Primer sequences for Pf869 and PgID19 were as published by Polley et al. [High throughput (HtLAMP) was performed in a 96-well microtitre plate as described , with soy et al.  except PPerformance of HtLAMP in a laboratory setting with limited resources involved storing reagents in a freezer at −20 °C and preparation of the mastermix as required for performance of each HtLAMP assay. Efforts to prevent contamination were based on physical separation of DNA extraction from mastermix preparation areas and spectrophotometry, combined with stringent attention to hand hygiene and glove and gown changes between the different assay areas.The limit of detection (LOD) for the HtLAMP assay was determined by performing HtLAMP-Pg and HtLAMP-Pf on duplicate samples at each dilution. Filter paper extracted using the two DNA extraction protocols and whole blood 5, 10 and 20 µL extracted using the two DNA extraction protocols were evaluated. The LOD was determined to be the lowest dilution  at which both duplicates were positive by HtLAMP.HNB is a metal ion indicator that is sensitive to changes in pH as well as concentrations of the divalent cations magnesium and calcium. Using pH calibration buffers at pH 4.0, 7.0 and 10.0 plus hydrochloric acid, 8 samples of different pH were produced and tested with HNB. Resulting colour changes were recorded visually. Magnesium sulphate (MW 246.68 g/mol) and calcium chloride (MW 110.99 g/mol) were used to make 5 samples of concentrations varying from 1 to 0.001 M. These were also tested with HNB and resulting colour changes were recorded visually.P. falciparum 3D7 DNA dilution series, HtLAMP mastermix reagents both with and without HNB were placed in the microtitre plate. Nuclease free water was used as the no template negative control. Following incubation in a water bath at 65 °C for 40 min, the microtitre plate was read visually to document cloudy and blue positive reactions and clear and purple negative reactions in the wells containing HNB. The presence and absence of cloudy white precipitate was recorded in the wells without HNB. The microtitre plate was then read in the spectrophotometer across the visible light spectrum from 300 to 700 nm to identify the wavelengths that best discriminated positive and negative samples both with and without HNB.A plate spectrophotometer (PowerwaveXS2 software version 2.00.18) was used in order to determine a threshold value to objectively differentiate a positive from a negative HtLAMP result and demonstrate the reliability of visually detectable colour change in determining a positive and negative sample. Using a The optical density (OD) value with the best discrimination of HNB related positive and negative colour change as well as presence and absence of magnesium pyrophosphate precipitate was then used to read each well. The threshold value for a positive reaction was calculated using the mean plus two standard deviations of the no template control (NTC) wells. This threshold value was then subtracted from the OD reading for each well to obtain a positive or negative numerical value. The presence of a visible blue colour change with presence of cloudy precipitate was mandatory for a sample to be positive. A positive numerical OD value was used to confirm the visually determined positive result. A sample that was purple and clear was considered a negative result with a negative OD value confirming this result. Samples with only a positive OD result without corresponding colour change and precipitate were considered negative. Therefore, results were recorded based on their colour change with OD values being used to confirm the visually detected result. Only samples with the appropriate colour change and corresponding OD result were considered true positives or true negatives for the purpose of statistical analysis.For both whole blood and filter paper DNA extraction methods, the limit of detection was determined by comparison of HtLAMP to nested PCR as described by Snounou et al. . PCR reaPCR was used as the reference standard for analysing the diagnostic accuracy of the HtLAMP platform given its sensitivity. Multiplex PCR (mPCR), which has an LOD of 0.02–0.05 parasites/µL as described by Padley et al. , and hadSensitivity and specificity of HtLAMP was calculated against mPCR and nested PCR. Sensitivity = true positives/ and specificity = true negatives/. The 95 % confidence intervals were calculated using an online calculation tool .The HtLAMP assay field applicability was assessed by performing the assay in a regional hospital in Kota Marudu district in Sabah, Malaysia. DNA was extracted from filter paper samples of 145 symptomatic patients using the FP-chelex rapid protocol, assayed using HtLAMP-Pg and HtLAMP-Pf and resulted were interpreted visually and confirmed with a portable spectrophotometer. Following a 3-day training workshop for local malaria staff on performance and interpretation of HtLAMP assay results, a questionnaire was completed on challenges of performing the HtLAMP assay in a regional laboratory setting.Plasmodium genus primers PgID19 or P. falciparum primers Pf869 [Plasmodium genus LAMP primers (HtLAMP-Pg) performed with a better analytical sensitivity than the P. falciparum LAMP primers (HtLAMP-Pf). For whole blood 10 and 20 µL, the chelex-saponin DNA extraction protocol resulted in better analytical sensitivity compared to using chelex alone. The explanation for the discrepancy in results for whole blood 5 µL for the two extraction protocols are unclear but may include pipetting error of blood at small volumes and partial loss of red cell pellet during the DNA extraction process. The best analytical sensitivity for HtLAMP-Pg was achieved using 10 µL of whole blood with the WB-chelex saponin protocol. This had a LOD of 2.5 parasites/µL which was equal to the nested PCR used. The best LOD for HtLAMP-Pf was 5 parasites/µL using 20 µL of whole blood with the WB-chelex saponin protocol. This was equal to the LOD for HtLAMP-Pg at this volume, while nested PCR had an LOD of 0.5 parasite/µL.The HtLAMP limit of detection was dependent on sample type, i.e. whole blood or filter paper, DNA extraction method and the LAMP primer used (rs Pf869 )  for HtLAMP-Pg, i.e. 2.5 parasites/µL compared to 25 parasites/µL. For filter paper samples, the LOD for HtLAMP-Pg using the FP-standard chelex protocol for DNA extraction was 2.5 parasites/µL, while the sensitivity of HtLAMP-Pg using the FP-rapid chelex protocol was 25 parasites/µL. The HtLAMP-Pf assay was able to detect 5 parasites/µL using the FP-standard chelex protocol and 50 parasites/µL using the FP-rapid chelex protocol. Therefore, the FP rapid chelex protocol appears to trade-off analytical sensitivity for a faster turnaround time for the assay. Taken together, these results confirm that the starting sample material and method of DNA processing have important effects on the analytical sensitivity of the HtLAMP assay.In the interest of improving turnaround time of the assay the FP- rapid chelex protocol was chosen despite a compromise in analytical sensitivity for the validation of the HtLAMP platform on filter paper samples from PNG and field testing in Sabah.When interrogated across the visible spectrum of light, wavelengths between 600 and 650 nm, appeared to best distinguish positive from negative samples, whether the cloudy magnesium pyrophosphate precipitate was present or not and whether there was a blue or purple colour change associated with HNB Fig. . The wavThe colour change associated with HNB was significantly affected by changes in pH, magnesium and calcium. At pH 3, HNB had a pink appearance compared with dark blue at pH 7 and sky blue at pH 10. Colour variation was also seen with magnesium, ranging from pink at a concentration of 0.001 M concentration to dark purple at a concentration of 1.0 M, and calcium, from dark pink at 0.01 M concentration to bright blue at 0.5 M concentrations. This suggests that maintaining the correct concentration of calcium, magnesium and pH of the mastermix, as well as awareness of other metal ions present during extractions processes, is important in allowing HNB to undergo its colour change during a LAMP reaction.P. falciparum. The features of each of these sample sets and composite sensitivity and specificity of HtLAMP-Pg/Pf for the detection of P. falciparum are outlined in Table Filter paper blood spots from symptomatic patients from Papua New Guinea and Sabah, Malaysia and whole blood samples from The Gambia were evaluated using HtLAMP-Pg and HtLAMP-Pf to determine the diagnostic accuracy of the assay for the detection of Plasmodium species. HtLAMP-Pf had a sensitivity of 97 %  and specificity of 96 %  for the detection of P. falciparum.Overall, HtLAMP-Pg had a sensitivity of 98 %  and specificity of 83 %  for the detection of Plasmodium species mono-infections was also assessed. In this context, the sensitivity of HtLAMP-Pg was 100 % , with a specificity of 83 % .The diagnostic accuracy of the combination of HtLAMP-Pg and HtLAMP-Pf for the detection of non-falciparum P. vivax positive) and specificity of 81 % (n = 13/16). There were 3 false positive HtLAMP-Pg results. HtLAMP-Pf was negative in all the mono-infection samples. For the 128 non-falciparum species infections from Sabah, HtLAMP-Pg had a sensitivity of 100 % (n = 128/128). HtLAMP-Pf was negative for 122 of these samples. For the 6 false positive HtLAMP-Pf results  there was no evidence of P. falciparum co-infection by PCR. In the Gambia samples, of the 95 HtLAMP-Pg positive samples, only 1 was negative by HtLAMP-Pf, suggesting a non-falciparum species mono-infection which was confirmed by PCR to be P. malariae with a parasitaemia of 7.5 parasites/µL.Subgroup analysis demonstrated that in the Papua New Guinea samples, HtLAMP-Pg had a sensitivity of 100 %  and sensitivity of HtLAMP-Pf was 88 %   with a 600 nm filter provided the optical densitometry values. Samples and HtLAMP reagents were stored at −20 °C in a freezer. Three separate benches within a large room were dedicated to DNA extraction, HtLAMP assay setup and spectrophotometry in order to prevent cross-contamination.The HtLAMP-Pg assay, performed in this resource-limited laboratory setting, had a sensitivity of 100 % . The HtLAMP-Pf assay had a sensitivity of 100 %  and a specificity of 95 % .Two local malaria laboratory staff was trained over a 3 day period to perform DNA extraction from filter paper and whole blood, setup the assay and interpret the results of the HtLAMP assay. Issues that were identified by local staff as potential limitations to deployment of HtLAMP in a field laboratory were difficulty of accurate sample placement into the 96-well plate and the availability and cost of reagents. However, it was felt that these limitations were balanced against the rapid turnaround time for the detection of parasites in microscopy negative samples thereby facilitating earlier patient treatment and household screening, potential time and cost saving in avoiding sending samples for PCR testing at reference laboratories and ease of interpretation of visual colour change results on the HtLAMP assay. The main challenges to setting up HtLAMP in this non-reference, low resource setting laboratory were ensuring good workflow to prevent contamination, and ensuring the reliability of ancillary equipment such as the centrifuge and freezers.To achieve its ambitious goals, the malaria elimination agenda will likely require diagnostic capacity to actively identify sub-patent infection in order to interrupt transmission. PCR, the most sensitive molecular diagnostic tool currently available, is limited by its technical requirements, particularly in resource limited field settings. A robust, low-cost and high-throughput assay for the rapid detection of malaria parasite DNA in finger-prick blood samples would provide this capacity. LAMP has been adapted into several different platforms , 42–44 iThe HtLAMP assay for the diagnosis of malaria that is described here has a simple platform—a standard 96-well microtitre plate, covered with a clear plate seal and incubated in a water bath at 65 °C, that is able to process a minimum of 85 samples . The use of a colorimetric indicator enhances visual detection of the assay endpoint. To improve objectivity of interpretation of results, the assay uses a spectrophotometer at 600 nm to produce a threshold value which correlates with the visible colour change. In addition, DNA extraction protocols for whole blood and filter paper based on chelex and saponin have been modified to improve turnaround time while maintaining the cost effectiveness of the assay.Plasmodium genus and species- specific primers may also enhance the LOD of the HtLAMP assay. Therefore, malaria elimination programs choosing diagnostic tools may be required to make important operational and logistics decisions with regards to both the type of assay used and the sample types, for example whole blood or filter paper, that can achieve the best analytical sensitivity for that assay in order to meet the current WHO target of 2 parasites/µL.However, there appears to be significant variation in the LOD of HtLAMP depending on sample type, DNA extraction method and primers selected, particularly so for filter paper samples where there was a ten-fold loss of analytical sensitivity using a DNA extraction method that had a faster turnaround time. Nevertheless, HtLAMP-Pg using 10 µL of whole blood with a chelex saponin protocol and HtLAMP-Pg on a 5 µL filter paper blood spot using the chelex overnight protocol achieved a LOD of 2.5 parasites/µL. This suggests that the HtLAMP platform has the potential to reach the WHO target LOD. However, further modifications, such as using commercial kits, optimisation of low cost DNA extraction methods or using a larger volume of blood are likely to be required to achieve the balance between improved analytical sensitivity and turnaround time. The availability of more sensitive LAMP primers to detect The HtLAMP assay has been validated in a resource limited laboratory setting using filter paper samples and the chelex rapid DNA extraction protocol, despite its aforementioned limitations, in order to evaluate its turnaround time in this clinical setting. Compared with PCR, which requires samples to be sent to a reference laboratory, HtLAMP-Pg had a sensitivity of 100 % and HtLAMP-Pf had a sensitivity of 100 % and a specificity of 95 % and results were available within 24 h.Plasmodium genus and P. falciparum on clinical samples from microscopy positive symptomatic and asymptomatic patients from regions with different Plasmodium species prevalence. Overall, HtLAMP-Pg had a sensitivity of 98 % and specificity of 83 % and HtLAMP-Pf had a sensitivity of 97 % and specificity of 96 % for the detection of P. falciparum. The use of the combination of Plasmodium genus and P. falciparum LAMP primers showed a HtLAMP-Pg sensitivity of 100 % and specificity 83 % compared to PCR for the detection of non-falciparum Plasmodium monoinfections. These results are comparable to other LAMP studies in symptomatic patients , the use of either crude or commercial DNA extraction protocols and the primer sets selected. Nevertheless, the cost per sample to perform HtLAMP-Pg on whole blood extracted using chelex-saponin protocol is USD$1. The turnaround time for completion of an assay, including DNA extraction, is 4–6 h.However, HtLAMP is not a quantitative assay, a limitation it shares with other LAMP based assays. Other potential limitations include the use of HNB, which is sensitive to changes in pH and metal ion concentrations. Awareness of, for example, the type of DNA extraction method used on samples, which may contain reagents that affect pH and metal ion concentrations, would help maintain the reliability of interpretation of the colour change aspect of the assay. Nevertheless, this is balanced against its low cost, stability at room temperature and easy visual interpretation of results. In its current format, HtLAMP relies on the storage of reagents at −20 °C. While an important issue in terms of field deployment, this could potentially be overcome by lyophilization of reagents.Plasmodium genus and P. falciparum primers, it could be adapted to detect any other blood-borne pathogen of interest, thereby broadening the potential applications of the platform beyond the diagnosis of malaria. Finally, HtLAMP has been performed successfully in a regional laboratory setting by local staff trained over a very short period of time demonstrating its ease of performance and interpretation of results.The HtLAMP platform offers several important improvements over currently available LAMP platforms. Firstly, HtLAMP is able to process up to 85 samples simultaneously which makes it an ideal potential platform for surveillance or household surveys. Secondly, it uses standard consumables, and can be performed using DNA extracted by chelex and saponin based protocols which makes it very cost effective. Thirdly, one of the significant advantages to the use of HNB is that it can be added to the start of the assay such that HtLAMP remains a closed system, thereby reducing the risk of cross-contamination. Fourthly, while the platform of HtLAMP has been used here with This provides confidence that the HtLAMP could be usefully deployed as a molecular diagnostic tool to support malaria elimination in resource-limited settings.


Disastrous effects and lifelong complications, ranging from respiratory and gastrointestinal burns to death can result from caustic soda ingestion. Accidental and non-accidental ingestions occur in different age groups. However, it is very troubling to find ingestion of caustic soda a very common occurrence among children below 5 years since they do not have the developmental level required to independently weigh up risks and are also under parental and societal protections. This study was therefore planned to investigate the ingestions of caustic soda by these children for purposes of proposing measures to curb the problem.Descriptive survey was employed for this study. A 14-item, semi-structure questionnaire was purposively issued to 57 parents/guardians whose wards had ingested caustic soda. Data was analysed with SPSS V.20.Twenty-seven (47.4 %) children got access to the soda at storage, 1 (1.86 %) was administered accidentally by a sibling while 29 (50.9 %) ingested during soap preparation. In respect of the former, the majority got access because it was stored in soft drink and water bottles in their parents/guardians rooms or kitchen. For the later, the children got access to the left-over soda because the soap-makers failed to adhere to good storage and disposal practices.Storage of caustic soda in soft drink and water bottles in accessible places, and training of children to drink directly from bottles influence caustic soda ingestion in children under five. Non-compliance to good practices of storage and disposal of caustic soda during soap preparation increases exposure and access of children to caustic soda ingestion. Ingestion of corrosive chemicals remains a common problem in the paediatric population and an important public health problem in many countries . GeneralThis phenomenon is disturbing because potentially catastrophic presentation and lifelong complications result from caustic ingestion , 7. GeneThe degree and extent of these complications or severity of damage depends on several factors such as concentration of caustic substance, dose or quantity swallowed, fullness of the stomach and duration of contact with tissue or organs , 13 and Daily Heritage with the heading; ‘Boy, 3 dying slowly after drinking caustic soda… family appeals for help’ [In Ghana, currently, caustic ingestions are causing severe morbidity and mortality in children and represent a worsening problem. In 2014, the Daily Graphic [or help’  has raisIn accordance with ethical considerations, this study was approved by the Ethics and Protocol Review Committee of the School of Allied Health Sciences, University of Ghana. During the period of January 2014 to April 2015, 57 patients within the age range of 1–5 years with histories of caustic soda ingestion underwent upper GI fluoroscopic examinations at the KBTH, a tertiary referral centre in Ghana. Because most hospital facilities in Ghana do not have functional fluoroscopy equipment, most of the caustic soda victims needing fluoroscopy evaluation of the oesophagus are usually referred to KBTH. Parents and guardians were contacted and invited after the fluoroscopic study of their wards to participate in the study. They were assured of privacy, confidentiality and offered the opportunity to withdraw from the study at any point in time. After consenting to participate in the study, they were then purposively issued with a 14-item, self-designed, semi structure questionnaire  of the children who ingested caustic soda agents were 2–3 years old  are shown in Table According to the respondents 27 (47.4 %) of the children got access to the soda at storage, 29 (50.9 %) during soap preparation process while 1 (1.86 %) was administered accidentally by a 10 year old sibling. For the latter the caustic soda was stored in a drug bottle and placed at the same window with other medicines prescribed for the victim. Upon the father’s instruction to administer the prescribed drugs, the victim’s sibling could not differentiate the drugs and subsequently administered the caustic soda as well.For respondents 27 (47.4 %) who indicated the children got access to the soda at storage, all of them said the soda was stored in soft drink and water bottles without proper fitting sealed covers, in their rooms or kitchen. For those who alleged their children accessed the caustic soda during soap preparation, 10 (34.5 %) confirmed the children got into contact with it when it was mixed with other materials and left in the open. A majority of 19 (65.5 %) respondents also said the children got into contact with the left-over soda and explained that soap makers preferred to finish the soap process before disposing of the left-over’s or waste.In respect of where the ingestion happened, 29 (50.9 %) of the respondents alleged the incident occurred during soap preparation at home, while the other 28 (49.1) supposed that the incident happened when their wards were sent to soap making family relatives or neighbours, for up-keeping. They further indicated that their wards drank the caustic soda because they felt it was water or drink.The majority 44 (77.2 %) of the respondents indicated they did not know much of the negative effects of caustic soda prior to their wards injury. Only 13 (22.8 %) of them admitted knowledge of the negative effects. All the respondents believed that most of the soap makers and other caustic soda users did not know much of the hazardous effects of caustic soda and hence made little efforts to properly secure it from children. All the parents/guardians also indicated that they trained their preschool children to drink directly from soft drink and water bottles.Ingestion of caustic soda often used for soap making in the traditional homes can cause death, and children who survive it may stay deformed with varying lifelong complications . This knChildren mainly got access to caustic soda at storage due to poor storage precautions, lackadaisical attitudes, and non-compliance with good practices on the part of traditional soap makers. The corrosive substances used for household soap were often stored in soft drink and water bottles without proper sealed covers, and placed in rooms and kitchens where children played. Eventually when the children saw these bottles they assumed or misidentified them as containing soft drinks or water and drank from them irrespective of the contents. This scenario which was previously reported by Adedeji et al.  in NigerIn other countries in different geographical locations, the incidence of corrosive oesophageal injuries has declined due to legislative efforts and stricter packaging standards , 17. UnfMoreover, it is apparent that 50.9 % of caustic soda ingestions occur during soap preparation process due to poor handling of left-over caustic soda. Soap makers often forget and do not put left-over caustic soda into hiding but always preferred to finish the soap process before. As a result, preschool children get access to these corrosive substances. The fact that the caustic substances are mixed with other substances and left in the open without proper safety measures also influences children access to and risk of ingestion.Only 1 out of 57 children was accidentally administered with the corrosive substance by a relative. The event to this action per the study is due to poor storage coupled with irresponsible parenting. The questions to ask are: How can caustic soda, a corrosive substance, be stored in a bottle and be placed at the same window where children drugs are also kept? And why should a parent instruct a 10 year old child to administer drugs to a preschool child? Obviously this mix-up where the caustic soda is accidentally administered is bound to happen.Children are to be protected by their parents or guardians and the society at all times. Therefore, it may suggest that these children with these kinds of accidental caustic ingestions are more likely to live in ‘unsafe homes or communities’ with regard to accessibility to toxic agents.In our study, the majority of children with caustic soda ingestions were in families whose parents did not have formal education, ply local soap making, undertake petty trading and farming as occupation. Unfortunately, the high costs of hospital admission, surgery, laboratory and radiologic examinations, coupled with the high cost of transportation to the referral centre for those living farther away are usually beyond the reach of many of these individuals resulting in late presentation of the injuries and further complications . FluorosSome researchers have also observed that repeated hospital visits also results in loss of work for parents and neglect of other children at home . Since mEffective legislation on caustic soda usage, educating parent/guardians on corrosive substances and further protecting children by the state are important measures that can be used to curb the onset of caustic soda ingestion in children below 5 years.Poor storage precautions where caustic soda substances are stored in soft drink and water bottles in accessible places and lackadaisical attitudes on the part of traditional soap makers, poor handling of left-over caustic soda during soap making and accidental administration of the corrosive substances influenced caustic soda ingestion in children below 5 years. Training of children to drink directly from bottles also influences caustic soda ingestion in children under 5 years. Corrosive ingestion is a considerable burden especially among people with little or no formal education and poor socio-economic status. This group of people should be targeted for education about creating safe working environments. Educating parents and soap makers will increase awareness of dangers associated with caustic soda and other corrosive substance ingestions. This is needful to guard against indiscriminate handing of these corrosive agents. Enforcement of chemical hazards legislation to control the usage of caustic soda is required to ensure that containers are child-proof to prevent easy access to corrosive agents by children.A suggested algorithm for preventing caustic soda ingestion in children under 5 years is presented in Appendix The study used relatively small sample size. Also, the study focused on a referral population to a tertiary hospital, so it did not include children with minor ingestions that were managed locally without referral.


Our understanding of cancer as a disease process has evolved tremendously over the centuries, culminating in the late 20th century with the discovery of oncogenes and tumor suppressor genes and subsequent understanding of carcinogenesis as it is depicted in the classic hallmarks of cancer paper by Hanahan and Weinberg . Genetic BRAF can be seen in unrelated cancers such as melanoma, colorectal and lung carcinomas [It is becoming clear that a given genetic alteration and associated molecular changes involving particular pathways in the neoplastic cell may not necessarily be specific for that particular type of cancer. Rather, such a genetic alteration represents a more general abnormality involved in the neoplastic transformation of a variety of cancers in different organs. For instance, mutations inrcinomas , 3, braircinomas , and hemrcinomas . This pa IDH-1 or IDH-2 mutations in diffuse gliomas, in contrast to well-circumscribed gliomas or reactive gliosis [Some of these alterations are also used as diagnostic aids in differential diagnostic settings, such as IDH-1 R132H identification by immunohistochemistry or the identification of other gliosis .An increasingly growing number of these alterations are now the subject of targeted therapies especially in the form of small molecule kinase inhibitors. They can also provide significant prognostic (such as FLT-3 mutation in acute myelogenous leukemia) and predictive information, further blurring the boundaries between diagnosis and treatment, as well as between basic and clinical sciences. It is not enough anymore for pathologists to provide only diagnosis but also an array of molecular markers that facilitate the discussion about prognosis for given cancer and potential therapeutic options.Of paramount importance are the explosion of knowledge in molecular biology and its clinical application in the form of molecular diagnostics, involving high-technology testing. Altogether, we have a better understanding of how such alterations operate in the process of oncogenesis, which in turn helps us better diagnose and treat neoplasms based on these alterations.6-methylguanine DNA methyltransferase (MGMT) gene methylation status in glioblastoma and response to alkylating agents [ KIT mutations in gastrointestinal stromal tumor (GIST) and response to imatinib [ ALK gene rearrangements in ALK-positive nonsmall cell lung carcinoma and response to crizotinib [These discoveries have also influenced the pharmaceutical and biotechnological fields, resulting in development of additional treatment options for cancer patients: Og agents , KIT mutimatinib , ALK genizotinib , and EGFizotinib  are a feDespite this enormous success over the last 50 years since the discovery of DNA double helix and the discovery of the first human oncogene, there are still a lot of questions in regard to the optimal way of molecular testing, distinguishing between passenger and driver mutations in a tumor, dealing with the vast intra- and intertumor heterogeneity, and introducing other nongenetic molecular markers such as proteins (proteomics) and metabolites (metabolomics). special issue, we present a variety of manuscripts that report technical, basic, and clinical research, molecular biology, and diagnostic and therapeutic aspects of neoplasia, as well as reviews of these subjects. The topics are not limited to a particular organ, system, or type of neoplasia. The manuscripts emphasize the importance of molecular markers in various aspects of neoplasia in an attempt to provide the reader with an up-to-date source of current research on molecular markers in cancer.In this


KRAS , BRAF , PIK3CA , KIT (commonly known as cKit) , ERBB2 (commonly known as HER2) (erb-b2 receptor tyrosine kinase 2), and EGFR  among others has been validated in gastrointestinal and pancreatobiliary tumors. However, the clinical utility of some of molecular biomarkers is still being investigated and validated. Although technically not a “molecular biomarker,” the utility of “molecular imaging” is being elucidated.The exponential increase in the use of molecular biomarkers as diagnostic, prognostic, and predictive aids in the management of cancer patients highlights the increasing importance of molecular biology in oncology. The clinical utility of some molecular biomarkers like MMP14 (previously known as MT1-MMP)  or previously known asmatrix metalloproteinase 14 (membrane-inserted)) and role in colorectal cancer, potential utility being described in other cancers [ SLC6A14 (solute carrier family 6 (amino acid transporter), member 14) and potential role in pancreatic cancer, potential utility in other cancers being described [This special issue covers some of the biomarkers currently in current clinical use and others being investigated, including the following: (i)escribed –7, (iii)escribed –14 of tuescribed –20.Michael O. IdowuJennifer LaudadioKathryn Rizzo


Heart failure (HF) is one of the leading causes for mortality and morbidity worldwide. Despite advantages in the management and treatment of this syndrome, nowadays, it is estimated that 50% of HF patients die within 5 years from diagnosis (McMurray et al., The present Research Topic aims to present some of the more relevant and recent acquisitions on the molecular abnormalities of the adrenergic system occurring in HF. Dr. Lymperopoulos has reported the molecular mechanisms of regulation of SNS in HF patho-physiology, discussing their therapeutic implications for the failing heart (Lymperopoulos, Evidences accumulated over the past 20 years support the pathogenic key role of cardiac GRK2 levels/activity in determining HF-related beta-adrenergic receptor dysfunction and cardiac inotropic reserve reduction. All these data indicate GRK2 inhibition, via gene therapy, as a new HF therapeutic approach that has been shown to be compatible and, in some models, also synergistic to beta-blockers. Cannavo et al. provide a contemporary update of this field by describing the therapeutic potentialities of this approach and its beneficial effects not only on beta-adrenergic receptor signaling, but also on cardiac metabolism, apoptosis, and mitochondrial dysfunction (Cannavo et al., The overall Research Topic indicates that the great advances achieved in the last decades in understanding the molecular alterations involved in the pathophysiology of HF are opening new opportunities for the treatment of this syndrome and, potentially, their future application to the clinical practice might result to further improvements of patient care. Moreover, the interesting new findings, discussed herein, will hopefully stimulate further research on these arguments.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The morphological and crystalline structural investigations reveal not only that the V-doped SrTiO3 photocatalyst possesses a uniform, porous, fibrous structure, but also that some V5+ ions are introduced into the SrTiO3 lattice. The photocatalytic capability of V-doped SrTiO3 porous nanofibers was evaluated through photodegrading methyl orange (MO) in aqueous solution under artificial UV–vis light. The results indicated that V-doped SrTiO3 porous nanofibers have excellent catalytic efficiency. Furthermore, the excellent catalytic activity was maintained even after five cycle tests, indicating that they have outstanding photocatalytic endurance. It is suggested that the excellent photocatalytic performance of doped SrTiO3 nanofibers is possibly attributed to the V5+ ion doping increasing the light utilization as well as to the outstanding porous features, the excellent component and structure stability.In this letter, we report a novel V-doped SrTiO Since Fujishima and Honda first reported photo-electrochemical water splitting using a TiO2 electrode [0 metal ions [4+, Zr4+, Ta5+, Nb5+ and V5+, as well as the development of new photocatalysts.Along with the rapid advancement of heavy industrialization, environmental pollution and excessive nonrenewable energy consumption have emerged as global issues –2. The mlectrode , many sttal ions –11, such3), an important multifunctional semiconductor, has been applied in photocatalysis technology for water splitting and organic contaminant degradation [3 is still heavily influenced by its considerably large band gap of ≈3.25 eV and high dielectric permittivity [3 shows that the top of the valence band (VB) and the bottom of the unoccupied conduction band (CB) are composed of the O 2p and Ti 3d-t2g states, respectively [3. Previous works showed that doping with 3d  and 4f  ions can significantly decrease the band gap through the hybridization of the Ti-3d and dopant-d states [3 also has an improved conductivity. Several groups have reported the excellent photocatalytic properties of Fe-doped SrTiO3, Nd-doped SrTiO3 and Ni/La co-doped SrTiO3 [3 nanomaterials.Strontium titanate  [3 nanofibers are stronger and integrally shifted to shorter angles compared to that of pure SrTiO3 nanofibers. The inset of 3 nanofibers are stronger than that of pure SrTiO3 nanofibers, meaning the former has better crystallinity. Using the Debye–Scherrer equation [3 porous nanofibers were calculated to be about 20.4 and 21.8 nm, respectively. As illustrated by TEM images shown in 3 nanofibers, which is consistent with the results from the SEM image. Moreover, 3. The average fringe spacing was measured to be about 1.42 Å which is larger than the 1.38 Å of the (220) plane of standard SrTiO3. Correlating these XRD results, it could be deduced that a few V ions were likely incorporated into the SrTiO3 lattice which then induced the expansion of the SrTiO3 lattice.The morphology and microstructure are very important for the development of an excellent photocatalyst. In 35-0734)  without equation , the ave3 porous nanofibers. All XPS data were corrected by reference to the C 1s peak at about 284.8 eV. As shown in 3 porous nanofibers. The atomic ratio of V/Sr/Ti is estimated to be about 1.9:48.6:49.5, meaning that the V ion concentration doped into SrTiO3 is 3.5 atom %. The high-resolution XPS spectra of O 1s  in aqueous solution under UV–vis light irradiation. In 3 porous nanofibers. No new absorption peaks were produced during the irradiation, which means that the MO is completely decomposed. C0: initial concentration of MO, C0−Ct: degraded concentration of MO at time t). This clearly demonstrates that only about 6% of the MO is degraded without any photocatalyst after irradiation for 75 min. In the case of the pure and V-doped SrTiO3 porous nanofibers, in contrast, the degraded MO is about 60% and >90%, respectively. Therefore, the self-photolysis of MO can be neglected. Furthermore, compared with pure SrTiO3 nanofibers, one can observe that porous V-doped SrTiO3 nanofibers exhibit an enhanced catalytic rate. Notably, the decrease of the MO concentration during the dark reaction could indicate that both of the two photocatalysts absorb the MO fluorescence well. 3 photocatalyst, revealing that pure SrTiO3 responds to UV light with absorption edge at about 388 nm but V-doped SrTiO3 nanofibers have a shifted absorption edge towards visible light. This means that V5+ ion doping improves the impurity level and narrows the band gap of SrTiO3, leading to the enhanced light absorption. Additionally, based on earlier research regarding 3d ion doping for SrTiO3, V5+ doping may also improve the conductivity of SrTiO3. Hence, the higher photocatalytic activity of V-doped SrTiO3 nanofibers compared to pure SrTiO3 nanofibers can be attributed to the V5+ ion doping. Stability and reusability are also very important for photocatalysts. As shown in the upper left inset in 3 nanofibers is barely changed after five catalytic reaction recycles, indicating the high photocatalytic endurance of the V-doped SrTiO3 nanofibers. This result was supported by the structure and component durability revealed from the SEM images . Secondly, V5+ ion doping provides an impurity level in the energy gap of SrTiO3 to improve the light response and conductivity. Thirdly, the V-doped SrTiO3 porous nanofibers also have considerable structure and phase stability. For a better understanding of the photocatalytic system of doped SrTiO3, more detailed investigations regarding V and other 3d ions co-doped into SrTiO3 are being carried out.In this work, it is suggested that there are three main contributors to the remarkable photocatalytic performance of V-doped SrTiO3 porous nanofibers were successfully synthesized via electrospinning followed by a thermal diffusion process. They were subsequently characterized by FESEM, TEM, XRD, XPS and UV–vis spectra in detail. During the photodisintegration of methyl orange (MO) under UV–vis light irradiation, the V-doped SrTiO3 porous nanofibers exhibited excellent photocatalytic activity. Furthermore, the cycling testing also confirms its outstanding catalytic endurance. The synergistic effect of V ion doping and the porous character is applied to understanding the excellent photocatalytic capability of V-doped SrTiO3 porous nanofibers. We believe that this work will benefit the fundamental research of 3d-ion-doped SrTiO3 as well as its photocatalysis applications.In conclusion, V-doped SrTiO3 nanofibers were prepared via electrospinning followed by heat treatment. 0.25 g of poly(vinylpyrrolidone)  and 0.34 g of Ti(C4H9O)4 were completely dissolved in a mixed solvent comprised of 0.6 g N,N-dimethylformamide (DMF), 1.0 g CH3COOH and 1.0 g C2H5OH under vigorous stirring. Meanwhile, a Sr(NO3)2 solution was also prepared by dissolving 0.147 g pf SrCO3 in 1.3 g of dilute HNO3 (15%). About 6 h later, a homogenous spinning emulsion with stoichiometric Sr2+ and Ti4+ was obtained by sufficiently mixing the above solutions. This was then transferred to a glass syringe with a stainless steel needle (inner diameter ≈0.4 mm) for electrospinning. The distance and voltage from the tip of needle to the collector were set at 20 cm and 17 kV, respectively. The feeding rate of the emulsion was set at 0.5 mL/h. After electrospinning, the as-spun nanofibers were annealed at 600 °C for 2 h with a heating rate of 2 °C/min. Then a portion of the resulting pure SrTiO3 nanofibers were immersed in a 20 g (1 wt %) NH4VO3 solution at 60 °C for overnight. Finally, the precipitates were separated, dried and annealed in air at 300 °C for another 2 h, and V-doped SrTiO3 porous nanofibers were then obtained.All chemical reagents were analytically pure and used without further purification. A typical preparation procedure as follows. Firstly, pure SrTiO3 porous nanofibers were characterized using field emission scanning electron microscopy  and transmission electron microscopy , X-ray diffraction , high-resolution transmission electron microscopy (HRTEM) and X-ray photoelectron spectroscopy  and UV–vis spectrophotometry (U-3600). The degradation of methyl orange (MO) in aqueous solution was used to evaluate the catalytic activity of the as-prepared photocatalysts. In a typical photocatalytic experiment, 30 mg of photocatalyst powder and 40 mL of MO solution (10 mg/L) were loaded together in a beaker (100 mL). The mixed solution was first stirred in the dark for 45 min to achieve an adsorption–desorption equilibrium between the dye and catalysts. The beaker was then exposed to a 175 W mercury lamp. During irradiation, an approximately 4.5 g suspension was sampled at regular 15 min intervals. The change in the MO concentration was monitored by recording the absorption peak maximum at about 464 nm of the MO via UV–vis spectrophotometry. After each catalytic reaction, the used photocatalysts powders were recycled by centrifuging at 3000 rpm and drying. They were then used for the next cycle. In this work, we performed five cycling tests to verify the catalytic endurance of V-doped SrTiO3 porous nanofibers.The surface morphology, crystal structure, chemical composition and optical properties of pure and V-doped SrTiO


The suggestion to start this Thematic Series was first made at the symposium held on September 16–18, 2014 in Potsdam, Germany, under the same title, “Molecular Machines and Devices.” The format of this Beilstein Nanotechnology Symposium is intended to bring a relatively small group of specialists together for a few days to discuss progress and open challenges. The range of topics is chosen such that it is not too wide in order to have enough concrete overlap of interests and backgrounds, but at the same time, wide enough to bring researchers together from fields that are somewhat disjoint. The three main topics in the 2014 Potsdam symposium were: molecular electronics, one-dimensional conductors, and synthetic molecular machines.Molecular electronics is currently being developed mostly at the interface between organic chemistry and nanophysics, leaning strongly towards the fundamental understanding of electron transport at the smallest scale and applications in nanoelectronics. The second topic, one-dimensional conductors, has been studied both theoretically and experimentally in the field of mesoscopic physics. More recently, truly one-dimensional wires, which are assembled from molecular building blocks into conductive oligomeric chains, have started to be investigated. This development provides a natural overlap between these two subfields of research. The third topic, synthetic molecular motors, has seen spectacular development in recent years, mostly in the research field of organic chemistry. In parallel, new theoretical ideas have been put forward by Tchavdar Todorov, Daniel Dundas, Mads Brandbyge, Per Hedegård, and others, which open the way for driving nanoscale motion by a dc current. The Potsdam meeting provided one of the first opportunities for researchers in each of these exciting but somewhat disconnected fields to interact and share ideas.The format worked out beautifully. The number of participants, the setting at the shore of the beautiful Griebnitzsee, the diversity of research themes, the combination of theory and experiment, physics and chemistry, and the quality of the oral contributions all contributed to an atmosphere of enthusiasm and creativity. This can be judged from the fact that discussions continued over lunch and dinner and until late evening between participants with widely different backgrounds. One of the intriguing problems discussed included magnetic effects such as the giant, low field magnetoresistance observed at room temperature in organic nanowires presented by Wilfred van der Wiel . The spiThis series presents the current research developments for many of these scientific problems and challenges. The works illustrate the interesting combination of theoretical questions, dedicated instrumentation development, synthetic chemistry, and experimental observations. Concepts for electrically actuated, molecular scale motion are discussed in many of the contributions, where switches, pumps, or motors are considered, and first experimental evidence is presented. The progress in theory and experiment on single-molecule electron transport can be judged from many contributions in this volume. On the other hand, some subjects are not as well-covered, including electron transport in biomolecules and one-dimensional conductors. This is merely the result of fortuitous fluctuations in having the proper material timely available for this volume, rather than a bias of interest.Indeed, important developments can be anticipated at the interface with biomolecular sciences. In parallel, the first applications of molecular electronics are appearing on the market in the form of molecular layers. The top-contact problem, which has hindered applications for a long time, is now at least partially resolved, so that we may anticipate more applications to follow. These will initially be in the form of layers of single molecules. The path to single-molecule devices is still long, but there is a lot of interesting material to be discovered on the way.Jan van RuitenbeekLeiden, February 2016


Spearman's rank correlations between FTICR-MS signal intensities of individual molecular formulae and optical properties determined which molecular formulae were associated with each PARAFAC component and optical index. The molecular families that tracked with the optical indices were generally in agreement with conventional biogeochemical interpretations. Therefore, although they represent only a small portion of the bulk DOM pool, absorbance, and fluorescence measurements appear to be appropriate proxies for the aquatic cycling of both optically-active and associated optically-inactive DOM in coastal wetlands.Optical properties are easy-to-measure proxies for dissolved organic matter (DOM) composition, source, and reactivity. However, the molecular signature of DOM associated with such optical parameters remains poorly defined. The Florida coastal Everglades is a subtropical wetland with diverse vegetation  and DOM sources . As such, the Everglades is an excellent model system from which to draw samples of diverse origin and composition to allow classically-defined optical properties to be linked to molecular properties of the DOM pool. We characterized a suite of seasonally- and spatially-collected DOM samples using optical measurements (EEM-PARAFAC, SUVA Dissolved organic matter (DOM) is an integral component of aquatic systems. DOM is fundamentally involved in many environmental processes, such as the binding of metals  are a quick and relatively inexpensive means for assessing DOM quality  offers detailed molecular-level information regarding the composition of DOM , and FTICR-MS. Spearman's rank correlations between molecular formulae assigned to mass spectral peak intensities and optical indices were obtained to determine which molecular families associated with different DOM optical properties. Molecular families which were found to be associated with multiple optical properties are also discussed.The Florida coastal Everglades is a subtropical wetland with diverse vegetation , salinity , and DOM sources . Wetland DOM dynamics are complex due to variable OM inputs, geomorphology, hydrology, primary production, and degradation processes , SRS4 (n = 12), SRS6 (n = 11), TS2 (n = 7), TS7 (n = 12), and TS10 (n = 10). Sites SRS2, SRS6, TS2, TS10 do not have samples representing all months. In these cases, the missing samples were either unavailable for analysis or not collected due to lack of water during the dry season. A complete list of sampling dates and exact locations are listed in Supplementary Table The Everglades is situated on the southern tip of the Florida peninsula where the regional climate is subtropical with distinct dry (November through April) and wet (May through October) seasons Lodge, . VegetatSRS4 n = 2, SRS6  is an indicator of DOM aromaticity and was defined as the absorbance at 254 nm (m−1) normalized to DOC  have been used as proxies for DOM molecular weight, photo-alteration, bio-alteration  260–455 nm at 5 nm intervals. Emission wavelengths were scanned from λex + 10 to λex + 250 nm  at 2 nm intervals. Individually-scanned spectra were then concatenated to generate EEMs. Post-acquisition data were blank-subtracted with Milli-Q water, absorbance data was used to correct for inner filter effects  were obtained using a Horiba Jovin Yvon SPEX Fluoromax-3 spectrofluorometer equipped with a 150 W continuous output Xe arc lamp in a 1 cm quartz cuvette . Mass spectra were calibrated using a reference mass list to achieve mass accuracies with an error less than 0.2 ppm. Molecular formulae consisting of C, H, O, N, S, and/or P were assigned to peaks with signal-to-noise ratios >5 according to published rules  and continuously infused into the electrospray ionization (ESI) source of a Bruker Solarix 15 T FTICR-MS instrument  at a flow rate of 120 uL hN = 0), saturated fatty acids , sugars (O/C = 0.9), and peptides . Since an individual formulae could occur in multiple isomeric structures, these classifications only serve as a guide to the structures present within DOM.Assigned molecular formulae were categorized by compound class according to elemental stoichiometries  were then obtained between normalized PARAFAC components, optical indices , and normalized mass peak intensities. Correlations between molecular formulae and optical parameters were considered significant at the 99% confidence level  were the most abundant, followed by CHOS  and polyphenols  also had considerable contributions to the overall molecular signature , saturated fatty acids  and sugars  contributed less to the molecular composition compared to other compound classes  vary with the source, molecular weight and the photochemical and biological processing of DOM , which suggests that C1 through C4 represented >80% of the molecular composition under the specific preparative and analytical conditions described for this study . Where EEM spectra are used to display the topographical distributions of fluorescence data, the molecular character of DOM revealed by FTICR-MS is facilitated by van Krevelen diagrams, where individual formulas are graphed according to their H/C and O/C ratios  and C2  represent “humic-like” components, which within the Everglades system are most likely terrestrially-sourced. Although C1 and C2 were both associated with high molecular weight, aromatic carbon-rich DOM  and CHON  formulae with considerable contributions from aromatic compounds, including black carbon and polyphenols  and polyphenols , and similar PARAFAC components for the FCE model  with increased contributions from N-, P-, or S-containing formulae, unsaturated aliphatics and peptides Table . These c254, S275−295, S350−400, SR, FI, freshness index, HIX) are listed in Supplementary Table r > 0, p < 0.01). The molecular composition of formulae positively correlated with each index is summarized in Table To expand on recent literature which primarily focused on how molecular formulae covaried with PARAFAC components alone . As such, similar molecular families, enriched in biolabile aliphatic formulae  were positively correlated with both S275−295 and SR , which in turn indicates low energy transitions  and SR(+) seem to be equally as effective in tracking the same pool of aliphatic-enriched compounds as the two PARAFAC components  which was not associated with any PARAFAC component  can provide nearly equivalent information regarding the molecular composition of DOM as PARAFAC modeling. However, it is important to note that such an observation can only be made for this particular suite of DOM samples using a four component PARAFAC model, which only offers coarse resolution of the EEM topography. The extraction of additional components may reveal more refined associations between molecular formulae and identified fluorophores for specific aquatic systems  was also observed in high latitude lakes (Kellerman et al., C/C peaks in lacustrine (Kellerman et al., C peak may serve as a proxy for black carbon due to the enrichment in condensed aromatic formulae which tracked with that particular component in boreal rivers. However, black carbon formulae tracked more closely with PARAFAC components exhibiting the AC+ peak in both the Everglades and lake environments (Kellerman et al., Conventional biogeochemical interpretations of PARAFAC components and other optical indices were generally in agreement with the bulk DOM molecular composition of formulae with which they were associated for the Everglades system. These patterns were also fairly consistent with previous research assessing optical and molecular linkages in other terrestrial aquatic systems (Stubbins et al., y Figure . RegardiOptical measurements, which reflect both the light-absorbing and light-emitting molecular components of DOM, are indicative of both the inherent optical properties of DOM compounds and how they interact with one another under specific environmental conditions. As such, DOM absorbance and fluorescence measurements are better described as emergent properties of the physico-chemical interactions among all organic and inorganic constituents which exist in the dissolved phase of a natural water sample. In the current study, the molecular families which tracked with optical indices were generally in agreement with conventional biogeochemical interpretations. In most cases, the optical indices broadly represented either terrestrial/allochthonous or microbial/autochthonous groups of DOM formulae. Although absorbance and fluorescence measurements represent a relatively small portion of bulk DOM pools, they have been validated here as informative proxies for the aquatic cycling of both optically-active and associated optically-inactive DOM in coastal wetlands and other aquatic environments.KC collected optical data and built the PARAFAC model and TD collected FTICR-MS data. SW interpreted the data and conducted statistical analyses with comments and suggestions from AS, RJ, and KC. SW wrote the manuscript with critical reviews and input from AS, RJ, KC, and TD.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.


FERMT1 (fermitin family member 1), and efforts to characterize genotype-phenotype correlations, the clinical variability of this genodermatosis is still poorly understood. In addition, several pathognomonic features of KS, not related to skin fragility such as aging, inflammation and cancer predisposition have been strongly associated with oxidative stress. Alterations of the cellular redox status have not been previously studied in KS. Here we explored the role of oxidative stress in the pathogenesis of this rare cutaneous disease.Kindler Syndrome (KS) is an autosomal recessive skin disorder characterized by skin blistering, photosensitivity, premature aging, and propensity to skin cancer. In spite of the knowledge underlying cause of this disease involving mutations of Patient-derived keratinocytes and their respective controls were cultured and classified according to their different mutations by PCR and western blot, the oxidative stress biomarkers were analyzed by spectrophotometry and qPCR and additionally redox biosensors experiments were also performed. The mitochondrial structure and functionality were analyzed by confocal microscopy and electron microscopy.Patient-derived keratinocytes showed altered levels of several oxidative stress biomarkers including MDA , GSSG/GSH ratio (oxidized and reduced glutathione) and GCL (gamma-glutamyl cysteine ligase) subunits. Electron microscopy analysis of both, KS skin biopsies and keratinocytes showed marked morphological mitochondrial abnormalities. Consistently, confocal microscopy studies of mitochondrial fluorescent probes confirmed the mitochondrial derangement. Imbalance of oxidative stress biomarkers together with abnormalities in the mitochondrial network and function are consistent with a pro-oxidant state.This is the first study to describe mitochondrial dysfunction and oxidative stress involvement in KS.The online version of this article (doi:10.1186/s13023-014-0211-8) contains supplementary material, which is available to authorized users. Kindler Syndrome , a rare heritable skin disorder with a complex phenotype and poorly understood pathogenesis, is characterized clinically by acral skin blisters in infancy and childhood, photosensitivity, and progressive poikiloderma [FERMT1 (fermitin family member 1) gene that encodes the protein kindlin-1, a component of focal adhesions in epithelial cells [KS results from recessive loss-of-function mutations in the al cells ,3. This al cells -7.FERMT1 have been identified as the origin of this disease, the complex phenotype of KS cannot be exclusively explained based on the adhesive function of kindlin-1. Therefore, the mechanisms responsible for clinical features such as photosensitivity and cancer are still awaiting to be unveiled [Although genetic mutations in unveiled . Severalunveiled . Reactivunveiled -11. To cunveiled . These iunveiled ,13. Glutunveiled ,15.In this study we sought to analyse at both, cellular and molecular levels, the potential derangements of the redox status in KS skin and keratinocytes. Using a variety of biochemical, molecular and morphological approaches we were able to detect an imbalance of oxidative stress biomarkers and mitochondrial abnormalities consistent with a pro-oxidant state in KS. Our results provide pathological bases for the non-adhesive clinical manifestations of this intriguing genodermatosis.Skin biopsies were taken from non-affected areas of KS patients’ arm, in which mutations, ages and gender are detailed in . Ultrathin sections (70 nm) were cut, stained with Reynolds’ lead citrate and examined under a Transmission Electron Microscope  using a digital camera . To identify the ultrastructural differences between patients’ skin specimens and controls, 10 randomized cells from 10 randomized areas of each cell culture were analyzed. Skin biopsies were obtained from control and KS patients and ten randomized mitochondria from eight keratinocytes were analyzed for each sample.FERMT1 gene. Polymerase Chain Reaction (PCR) amplification of FERMT1 gene was performed on genomic DNA as previously described [Intronic primer pairs were designed to amplify individual exons and flanking splice sites of the escribed ,16. PCR 2 and the culture medium was changed every other day. Third to fifth passages cells were used as indicated for all experiments.Skin biopsies were incubated two hours at room temperature with collagenase (Sigma) ). Detached epidermal sheet was then incubated with tryspin solution (Sigma) for 20 minutes at 37°C (four cycles of trypsin were done). The released keratinocytes were centrifuged at 1000 rpm for 7 minutes ,18. The Lipid peroxides were determined by measuring MDA, which is formed from such peroxides. MDA from the samples reacted with thiobarbituric acid (TBA) at 100°C to form a MDA-TBA adduct. The protein-free extract was separated by HPLC (Ultimate 3000 Bionex) on a column of octadecyl silica gel  to separate the MDA-TBA adduct from interfering chromogens. The adduct was eluted from the column with 50 mM phosphate buffer pH 6.8- methanol 50% and quantified spectrophotometrically at 532 nm.GSH and GSSG levels were studied using the Glutathione Fluorescent Detection Kit  following the instructions of manufacturers for cellular material. Briefly, cells were prepared in 5% sulphosalicilic acid and centrifuged at 13,000 g to separate the proteins. The supernatant containing the GSH and GSSG was reacted with ThioStarreagent to produce a fluorescent product . Addition to the sample of a reaction mixture containing NADPH and GSH reductase converts all the GSSG into free GSH, which then reacts with ThioStar, yielding the signal corresponding to total GSH. The difference between both measures offers the amount of GSSG.Total RNA was isolated from cells using the PARISTM Protein and RNA Isolation System  according to the manufacturer’s instructions. For reverse transcription reactions (RT), 1 μg of the purified RNA was reverse transcribed using random hexamers with the High-Capacity cDNA Archive kit  according to the manufacturer’s instructions. RT conditions comprised an initial incubation step at 25°C for 10 min to allow random hexamers annealing, followed by cDNA synthesis at 37°C for 120 min, and a final inactivation step for 5 min at 95°C. The mRNA levels were determined by quantitative real-time PCR analysis using an ABI Prism 7900 HT Fast Real-Time PCR System . Gene-specific primer pairs and probes for GCLC and GCLM , were used together with 1x TaqMan® Universal PCR Master Mix  and 2 μL of reverse transcribed sample RNA in 20 μL reaction volumes. PCR conditions were 10 min at 95°C for enzyme activation, followed by 40 two-step cycles . The levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression were measured in all samples to normalize gene expression for sample-to-sample differences in RNA input, RNA quality and reverse transcription efficiency. Each sample was analyzed in triplicate, and the expression was calculated according to the 2-ΔΔCt method .Neurospora crassa ATP synthase protein 9 (mito-Grx1-roGFP2) determines the glutathione redox potential in mitochondria.Glutaredoxin-1 (Grx1) is an enzyme that specifically catalyzes the equilibrium between the redox pair of interest, reduced glutathione (GSH) and oxidized glutathione (GSSG) in the cytoplasm. In this way, Grx1-roGFP2 fusion protein allowed dynamic live imaging of the glutathione redox potential in the cytoplasm with high sensitivity (from millimolar to nanomolar changes) and temporal resolution, facilitating the observation of physiologically relevant redox-based signals responding to exogenously applied agents ,24. The Retroviral supernatants from PA317 Grx1-roGFP2 and mito-Grx1-roGFP2 lines  were collected and used for control and patients human keratinocytes infections (three controls and three patients). Two rounds of seven hours infections were performed and keratinocytes were grown until confluence with keratinocyte medium (percentage of infection was higher than 75%). GFP positive cells were analyzed by flow cytometry  either in basal state or after addition of 12.5 μM hydrogen peroxide solution. Cells were excited with 405 and 488 nm lasers and the ratio of emissions in the green channel was calculated. Flow cytometry data were analysed using FlowJo version 7.6.1.Keratinocytes were lysed with a lysis buffer prepared with Tris pH 7.5 50 mM, NaCl 150 mM, Triton 1.5%, EDTA 1 mM, protease inhibitor cocktail tablets (Roche), ortovanadate, sodium pyrophosphate and sodium fluoride. The lysates were then loaded with LDS Sample Buffer (Invitrogen) or manufactured sample buffer 5X and run on NuPage 4–12% Bis-Tris gels (Invitrogen) at 120 V for 2 hours for antioxidant enzymes. The proteins were then transferred onto nitrocellulose membranes (Invitrogen) and blocked in 5% skimmed milk in 0.1% Tween-20 (Sigma-Aldrich) and phosphate-buffered saline. The membranes were then probed with anti-kindlin1 (1:10.000) antibody  overnighThe mitochondrial distribution and morphology was studied by confocal microscopy. Cells were plated in rounded glass in Cnt-BM.1 medium and after 48 h of culture they were stained to localize mitochondria and nuclei. Cells were loaded with Mito Tracker Red™ (Invitrogen) in a final concentration of 250 nM in cell culture medium, 37°C and 5% CO2 and after 30 minutes of incubation they were mounted with DAPI (Molecular probes). The fluorescence was detected by confocal microscopy ) and the plane  that had most mitochondria was captured. Images are representative examples of 3 separate experiments.Mitocondrial’s membrane potential was determined with JC-1™ vital dye (Invitrogen). It is a cationic dye that exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (~520 nm) to red (~590 nm). Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. Cells were plated in rounded glass in Cnt-BM.1 medium and after 48 h of culture they were stained with 2.5 μg/mL JC-1™, 10 minutes, 37°C. After that, glasses were mounted with DAPI and pictures were obtained with a confocal microscope.For the statistical analysis of the results, the mean was taken as the measurement of the main tendency, while standard deviation was taken as the dispersion measurement. A Student-Newman-Keuls method was used to determine the significance of differences, when analyzing GSSG/GSH ratio, GCLC and GCLM relative expression determined by qRT-PCR, MDA levels studied by HPLC-UV, biosensors by flow cytometry and JC-1 intensity by confocal microscopy. The significance has been considered at *p < 0.05, **p < 0.01 and ***p < 0.001, as indicated in each case. GraphPad Software v5.0 was used for statistical analysis and graphic representations.Three unrelated patients with KS were studied. The patients’ mutations, age and gender are summarized in Additional file These patients represent a proper sampling of the spectra of mutations found in KS, with one patient showing total lack of expression (SK2), one presenting a truncated protein (SK1) and a third expressing a normal size protein with a single amino acid substitution and reduced expression (SK3).GSH is one of the most important protective mechanisms against ROS. In this regard, one of the best-characterized biomarkers of cellular oxidative stress is the ratio between oxidized and reduced glutathione (GSSG/GSH) -15. The We also studied another marker of oxidative stress, malondialdehyde (MDA). This is a product of the degradation of polyunsaturated lipids by ROS . Our res. KS and control keratinocytes expressing Grx1-roGFP2 or mito-Grx1-GFP2 were treated with hydrogen peroxide solution (H2O2) and analyzed by flow cytometry. The basal biosensor levels in the KS keratinocytes (in the absence of H2O2) already showed a higher oxidized/reduced ratio than their respective controls. After H2O2 challenge, the same tendency was observed, indicating a higher pro-oxidative state in KS keratinocytes . A second sensor containing a signal peptide directed to the mitochondria (mito-Grx1-roGFP2) was also used to detect oxidative stress in the mitochondrial matrix-roGFP2. We sought to determine whether deranged redox status could translate into ultrastructural changes in target organelles. For this purpose, the morphology of mitochondria in patient skin biopsies and cultured keratinocytes was analyzed by electron microscopy. The Figure The natural history of a genetic disease is not completely described by the simple identification of the causal mutation but requires also the understanding of biochemical and molecular mechanisms that are directly responsible of the phenotypic characteristics of the disease. Sometimes this is challenging given the multiple interactions of genes downstream of the mutation. This appears to be the case of KS for which several aspects of its pathogenesis remain uncertain. Since two of the unexplained features of KS, photosensitivity and cancer development, have been associated with oxidative stress, we have advanced the hypothesis that some clinical features of KS patients may be related to a redox imbalance and higher susceptibility to oxidative stress.Our results cover mutation spectra and phenotype since they are based on three very different types of mutations. They include a simple hypomorphic mutation caused by a single amino acid substitution (SK3), a mutation resulting in a truncated protein (SK1), and a patient null for kindlin-1 (SK2). Furthermore, given the importance of age in the symptomatology of this disease, the three patients studied reflect different ages, two children (SK1 and SK2) and one adult (SK3). For redox studies, we also took into account the patients’ gender  and consequently, in every case the control was matched by age and gender. As for other genodermatoses, availability of samples to derive keratinocyte cultures is usually low. In spite of this drawback, our results, obtained with primary patient cells covering different mutations and ages, follow a clear trend regarding oxidative status and mitochondrial alterations.The oxidative stress biomarker analysis revealed that GSSG/GSH ratio was higher in SK1 and SK3 keratinocytes (Figure Cytoplasmic and mitochondrial abnormalities in redox balance confirmed by Grx1-roGFP2 biosensors system, in KS keratinocytes (Figure Taken together, our results suggest that alterations of the redox balance in KS may be a potential explanation of the premature skin ageing and cancer prone phenotype of these patients.As discussed above, our results with the biosensor probes point to the cytoplasm and the mitochondria as potential sources of ROS. For this reason, we decided to study morphological and functional alterations of the mitochondria of KS patients. Electron microscopy of both, skin biopsies and cultured keratinocytes from KS patients showed morphological alterations which are consistent with dysfunctional mitochondria Figure  as previTo our best knowledge, neither oxidative stress nor alterations in the mitochondria have been previously reported in KS. It is not yet clear whether a derangement of the redox status in KS cells is cause or consequence to mitochondrial dysfunction and morphological aberrations. The most likely explanation is that mitochondria are both the cause and effect of the oxidative stress. Therefore, we envisage a vicious circle where ROS-altered mitochondria results in increased mitochondrial oxidative stress.Fermt1 knock out mouse model associated kindlin-1 deficiency to αv β6 integrin-mediated increase of TGFβ activation [At this point it is not clear how alterations in kindlin-1 may lead to generation of oxidative stress. It is unlikely that the pure disruption of the kindlin-1 synthesis would explain this phenomenon. A likely explanation is that alterations in kindlin-1 function lead to a disruption in the signal transduction pathways involving integrins and focal adhesions -40. In ftivation . It is tOur results suggest that KS keratinocytes are cells under severe oxidative stress, a condition which could underlie some of the obscure aspects of the disease such as photoaging, photosensitivity and ultimate, the high risk of cancer development.