Pseudomonas aeruginosa and increased resistance to infection in burn patients recommend the issue of infection control. In this research, we study ZOUSH herbal ointment for gene silencing of Pseudomonas aeruginosa.The outbreak of MDR and XDR strains of Satureja khuzestaniea, Zataria multiflora, Mentha Mozaffariani Jamzad, honey, and polyurethane. The MIC and disk diffusion tests were examined by single, binary, tertiary and five compounds.The herbal ZOUSH ointment was formulated by alcoholic extracts of plants Pseudomonas aeruginosa. During the interval of 5 days, cultures were done from the liver, blood, and wound by four consecutive quarters and counting of Pseudomonas aeruginosa was reported in the liver.Three-week-old mice were considered to be second-degree infections by In this study, silver sulfadiazine ointments and Akbar were used as a positive control. The gene gyrA reference was used as the control. Real-time RT-PCR results were evaluated based on Livak as the comparative Ct method.exoS, lasA, and lasB after treatment with ZOUSH ointment. SPSS Analyses showed that reduction of expressions in genes exoS, lasA and lasB after treatment with ZOUSH ointment were significantly meaningful (p < 0.05).The In vitro results indicated that wound infection was improved by healing wound size in the treatment groups compared to control treatment group. In this research, the changes in gene expression were evaluated by molecular technique Real-time RT-PCR. The results showed downregulation Pseudomonas aeruginosa in the mouse model with the second-degree burn. The positive effects decreased in the number of bacteria by reducing the expression of virulence bacteria genes as exoS, lasA and lasB and improvement of wound healing.Our study showed that ZOUSH ointment has the positive effect for gene silencing  Pseudomonas aeruginosa is an opportunistic gram-negative bacil. When it is entered into a host cell, its employee several virulence factors such as cell-associated and extracellular factors [ factors .This opportunistic bacterium is responsible for severe and fatal infections in patients with underlying diseases, such as Cystic fibrosis, chronic respiratory infections, Bronchiectasis, Severe Burns, Neoplasia, Neutropenia, Diabetes, AIDS and other diseases –4.Pseudomonas infection [This bacterium is a common cause of nosocomial and burn wound infections . Over thnfection . Each yenfection . In the nfection .Candida albicans, 20–30% for P. aeruginosa and 5% for Staphylococcus aureus [According to conducted research, the mortality rate reported 30–50% for s aureus .One of the most important side effects of burns is the infection which is caused by burn and war wounds. Dau and et al. reported that 7% of war wounds were related by burn wounds . On the Zataria multiflora and Satureja khuzestanica not only have many applications in traditional medicine but also they have phenolic components like thymol and carvacrol which have antimicrobial activities against fungi [Some plants such as st fungi , 12.P. aeruginosa to many antibiotics, the treatment should be selected based on the antibiotic susceptibility of the strain because resistant strains can quickly emerge during treatment. In addition to, certain clinical infections caused by P. aeruginosa should not be treated with one drug because the success rate of such treatments is low, and the possibility of developing drug resistance increase [Due to the resistance of increase .P. aeruginosa to broad-spectrum penicillins, some cephalosporins, carbapenems, monobactams, fluoroquinolones, aminoglycosides, and colistins [P.aeruginosa isolates showed multiple-antibiotic resistance with rate 50% in Europe and about 35% in the United States [The presence of the efflux pump systems and β-lactamases contribute to the intrinsic resistance of olistins . The posd States , 16.Iran with 11 different climates and more than 7500 plant species is regarded an excellent region to achieve valuable and rare medicinal species. Currently, 25% of the existing drugs are made from herbal sources, and 12% of the drugs are made from microbial sources .Zataria multiflora Boiss are belonging to the family Labiatae with at least 6% of essential oil (volume/weight) [Thyme, dried flowering plants, and /weight) –19.Origanum vulgare L.) from the family Lamiaceae has at least 0.1% (volume/weight) essential oil [Satureja khuzestanica is an herbaceous, aromatic and perennial herb of the family Lamiaceae with numerous branches. Satureja is one of the genera of the family Lamiaceae belonging to the subfamily Nepetoideae and the tribe Mentheae [Oregano  in Kashan Barij Essen [Alcoholic extracts of ij Essen .S. khuzestanica, Z. multiflora, and O. volgarum extractions, Honey, and Polyurethane with the equal ratio [The extracts were dissolved in 1/3 dimethyl sulfoxide, Honey was dissolved in about 1/3 of distilled sterile water and The Polyurethane was first dissolved in 6 M urea at a rate of 1/3 and then placed on indirect heat for evaporation of urea. This solution was prepared as a stock. The ZOUSH ointment is prepared with the compounds of al ratio .The inhibitory effect of the ointment and its base is determined by disc diffusion method and then compared with the reference antibiotic .Pseudomonas aeruginosa PAO1 was prepared in Pseudomonas agar medium.Twenty-four-hour culture of The stock of 0.5 McFarland (according to CLSI 2016) was prepared as a control in a separate test tube for opacity comparisons.Pseudomonas aeruginosa PAO1 was inoculated into the physiological serum.Colonies from the 24-h culture of 8 CFU.In addition to the 0.5 McFarland protocol, the method of measuring the OD in the tubes at 400 nm wavelengths and number of bacteria calculated based on CFU. Absorption of these samples with a spectrophotometer, in a wavelength of 625 nm, would be 0.08–0.13 that contains about 1.5 × 10Pseudomonas aeruginosa and the appropriate concentrations of the original stokes of ZOUSH ointment  was inoculated. The proper concentration of primary stock: 5–10–20-40-80-160-320).In seven tubes, 2 ml of MHB broth medium, 10 μl of The tubes were incubated for 24–18 h in a 37 °C.The incubation tubes were cultured on an MHA agar medium and then incubated at 37 °C for 18–24 h.At the end of the incubation, time MIC was determined.MIC was calculated for each extract single, binary, a triple mixture, as well as a primary stock (ZOUSH ointment compounds).The following steps were taken to determine the MIC:50 value is determined to calculate the number of bacteria should be inoculated to the burned mouse, and after 5 min the bacteria are inoculated on the back surface of the mouse [The LDhe mouse .The mice were classified into 5 groups. These groups and various treatments had shown in Table Burns are induced on the back surface of the BALB/c mice according to Ian Alan Holder method .First, the mice were anesthetized by intraperitoneal injection of xylazine-ketamine anesthetics. 9.5 cc ketamine was mixed with 0.5 cc xylazine and is used as stock of anesthetics. 0.1 cc was injected for every mouse.2.Dorsal surface hair was removed using a cream in a dimension of 3 × 3 cmThe back of the body was disinfected with 70% alcohol.2 in size, and then sprayed with 96% alcohol, and then the flame was placed on the back surface of the mouse-free hair. Thus, 30% of the skin of the animal suffered from grade 2 burns.Aluminum pieces were pre-designed in 2 × 2 cmTo prevent dehydration of the animal, 0.5 ml of normal saline was injected subcutaneously into the animal’s back.Five minutes after burning the animal was injected with 20 μl suspension with a sub-lethal half-Mcfarland concentration at the site of the wound and then spread.The mice used in this study were female BALB /c mouse old 6–9 weeks. In order to make the second degree of burns, Ian Alan Holder method as following steps carried out.The treatment period of the mouse was started 24 h after burning. Treatment of each group was performed according to the schedule and was performed as two times in the morning and afternoon at certain times. Also, the amount of ointment used was the same for each mouse.S. khuzestanica, Z. multiflora and O. volgarum, Honey and Polyurethane. The active ingredient natural Akbar 1 Ointment was Onosma dichroanthum Boiss, Ricinus communis, the essential oil of Mentha and Z. multiflora. Also, the active ingredient is silver sulfadiazine, silversmith (10%) and synthetic sulfadiazine (1%) of the sulfonamide group [The active ingredients of ZOUSH Natural Ointment include de group , 31.As much as a standard loop, blood was cultured from the animal’s heart on a medium of Blood agar.At the end of the phase, the rat liver was removed and inserted into microtubes.The liver was weighed and its average was about 1 g per mouse.In the next step, 100 μl serum sterilized physiology with supplements was added to the microtube and pipetted.Then, 10 μl of microtube solution was cultured on Blood Agar.The cultured plates (blood and liver) were incubated for 24 h at 37 °C. , 33.The specimens were prepared from each group, in fifth , tenth , and fifteenth and twentieth days after treatment, the number of mice was selected for microbiological, Pathology and biochemical studies. The following operations were performed for each mouse:First sampling was done with sterile swabs from the wounds. Then, the swab was placed inside sterile tubes containing 0.5 ml of physiological saline then culture carried out on Blood Agar. The cultured samples were incubated for 18–24 h at 37 °C. Then, the colonies examined by morphological and biochemical tests to determine the identity of isolated strains , 35.lasA and exoS genes and housekeeping gene gyrA were investigated using Real-time RT PCR technique. [The inhibitory effects of ZOUSH ointment with MIC concentration on chnique. , 36, 37The inhibitory effect of ZOUSH ointment with MIC concentration on the internal housekeeping DNA Gyrase A is investigated using Real-time RT PCR. Statistical analyses were carried out by descriptive and statistical methods (SPSS 24).P. aeruginosa PAO1 using broth microdilution and broth microdilution techniques according to CLSI 2016.Table The results showed that the ZOUSH ointment compounds had better and greater antibacterial effects compared to any of the substances as single, dual mix and triple mix. On the other hand, since the extractions were color combinations, so all tubes were cultured on Muller Hinton agar to determine the MIC value using broth microdilution and broth microdilution techniques; the first non-growth plate was considered as MIC.p values < 0.05 that indicated to be meaningful.The results of the table demonstrated that the MIC value of the ZOUSH ointment mixture has decreased, indicating better and greater antibacterial effects of the ZOUSH ointment combinations compared to any of the substance as single, dual mix and triple mix. Result mean in per reaction was calculated. Results analyzed with SPSS version 24 with P. aeruginosa PAO1, the antibiogram test was performed for Gentamycin (10 μg), Imipenem (10 μg), Meropenem(10 μg), Polymyxin B (300 u) and Tobramycin(10 μg) according to CLSI 2016 by Kirby-Bauer method that P. aeuginosa was sensitive to all antibiotics.To determine the resistance and sensitivity of P. aeruginosa PAO1, the antibiogram test was performed using the discs contain mixed extracts as single, dual and triple mix as well as primary seed stock mix  with different concentrations. The results indicated Disc diffusion with mixed compounds with Honey, Mentha Mozaffariani Jamzad, Satureja khuzestaniea and Zataria multiflora with concentrations 10, 13, 17 and 20 μg were 12, 14, 15 and 18 mm respectively.In order to determine the sensitivity of Four mice were randomly excluded from each group at intervals of 5 days and on days 5, 10, 15 and 20 after treatment and were killed for microbiological, pathological and biochemical studies. The results of blood and liver cultures at different phases are reported as follows:P. aeruginosa was inoculated into the burn wound, but in addition to P. aeruginosa, S. aureus was isolated from blood and liver cultures of different mice groups.In the phase of burning according to Ian Alan Holder method, only Staphylococcus strains such as S. aureus are known as normal flora of the primates and mice body . Therefore, isolated S. aureus was not counted.P. aeruginosa isolated from the liver cultures in the exclusions 1 to 4 at intervals of 5 days were presented in the following 5 based on CFU/g. First exclusion of mice was done 5 days after treatment, second exclusion 10 days after treatment, third exclusion 15 days after treatment, and fourth exclusion  20 days after treatment  presents automatically all data on the Real-time RT-PCR reaction after each test and saves as an eds file. Each eds file contains the raw graph of the reaction progress in exponential mode. This file involves the test results as Amplification plot, Multicomponent plot, Melt curve, QC summery, Row data plot and Multiplot view. Amplification plot of Real-time RT-PCR reaction for For each sample, Real-time RT-PCR reaction was performed before and after treatment. Meanwhile, only one diagram of each reaction is given in below and no other reaction information is provided because of similarity Fig. .For each sample, eight Real-time RT-PCR reactions were performed before and after treatment. Meanwhile, only one diagram of each reaction is given in below and no other reaction information is provided because of similarity Fig. .For each sample, eight Real-time RT-PCR reactions were performed before and after treatment. Meanwhile, only one diagram of each reaction is given in below and no other reaction information is provided because of similarity Fig. .The relative quantification method measures relative changes in the expression levels of a gene mRNA relative to a housekeeping gene mRNA. In this method, unlike absolute quantification, there is no need to a standard curve, and the housekeeping genes that have stable expression are measured as reference genes.2-ΔΔCt or comparative Ct method, is performed without modifying its efficacy, and it is assumed that the desired doubling of target and reference DNA genes runs throughout each cycle.The relative calculation of the gene expression according to the Livak method (ΔΔCt method), also known as lasA gene and 1.07 fold for the lasB gene. The resulting analysis by SPSS version 22 demonstrated relation gene expression before and after treatment is meaningful (sig = 0).Given the information, such as Ct, obtained from the amplification curve of the genes studied in the Real-Time RT-PCR method, the mean ΔCt before and after treatment was determined for each of the genes; subsequently, Cts were normalized. Figure Pseudomonas aeruginosa is an important opportunistic gram-negative bacterium that causes high mortality in immunocompromised patients. This bacterium is the third agent of hospital-acquired infection and the second pathogen of burn wounds [n wounds .Burn is one of the most serious medical conditions, which affects whole physical and mental aspects, and is capable of infecting people of all ages .P. aeruginosa spread in hospital settings is difficult due to the intrinsic and acquired resistance of this bacterium to many antibiotics. Despite many scientific advances in the treatment of burns, it remains one of the major public health problems around the world, especially in developing countries [Prevention of ountries , 41.P. aeruginosa [Traditional medicine plays an important role in the treatment and prevention of these bacteria. The efficient method is needed for immediate treatment of this bacterium due to therapeutic dilemmas, drug resistance and high mortality due to ruginosa .S. khuzestaniea extract, which inhibits the ATPase activity and increases the bacterial cell membrane permeability, and hence enhances the membrane permeability of antibacterial agents [Carvacrol is a major component of the l agents .S. khuzestaniea [Farzaneh et al. determined that carvacrol, γ-terpinene, and p-cymene constitute the major components of estaniea .S. khuzestaniea on P. aeruginosa [Esmaeili et al. reported the inhibitory effect of ruginosa .Qing-huan Yin et al. examined the anti-inflammatory and apoptotic effects of carvacrol on hepatocellular carcinoma cell line HepG-2 and observed its anticancer effect .Listeria monocytogenes at 37 °C but was effective at 55 °C [S. khuzestaniea essential oil on clinical isolates of Escherichia coli [S. khuzestaniea and Z. multiflora with broth microdilution method on E. coli, S. aureus, C. albicans and Aspergillus niger [S. khuzestaniea was 80 μg/ml for P. aeruginosa [Kamdem et al. showed that carvacrol had no specific effect on at 55 °C . Mahbubihia coli . Jourabius niger . In the ruginosa .S. khuzestaniea was reported 0.5 μg/ml for P. aeruginosa [S. khuzestaniea.In the study of Jalalvand et al., the MIC of ruginosa ; but in S. khuzestaniea were examined on infectious bacteria in the respiratory tract [In a study by Amiri et al., the antibacterial properties of ry tract .S. khuzestaniea was performed on C. albicans, E. coli, S. epidermidis and B. cereus using disc diffusion and broth microdilution techniques [In a study by Ghodrati et al., the antibacterial effect of chniques .In studies conducted by investigators, antibacterial effects these plants reported –39.P. aeruginosa was decreased, but this rate of decline was much lower than the rate of treatment with ZOUSH ointment. The mean count of isolated P. aeruginosa in treatment with Akbar 1 ointment after 20 days of treatment was equal to 10 days of treatment with ZOUSH ointment. These results indicate the bactericidal feature of the ZOUSH ointment had nearly two-fold higher effects.In the group treated with Akbar 1 ointment, the mean count of isolated P. aeruginosa in the exclusion 2 had a significant increase compared to the exclusion 1, and two mice died in this group between the exclusions 1 and 2, due to the sudden increase in the mean count of the bacteria, and thus inability of the body. The silver sulfadiazine ointment was used to control the infection, which eventually occurred death. Moreover, the mean count of isolated P. aeruginosa in treatment with silver sulfadiazine ointment was decreased since the 10th day of the treatment. However, the bactericidal feature in the group treated with ZOUSH ointment was much higher, so that the mean count of isolated P. aeruginosa after 15 days of treatment with silver sulfadiazine ointment was even higher than the mean count of isolated P. aeruginosa after 5 days of treatment with ZOUSH ointment. These results indicated the bactericidal feature of ZOUSH ointment with an effect three times higher than to silver sulfadiazine ointment.In the group treated with silver sulfadiazine ointment, the mean count of isolated P. aeruginosa not only was reduced but also was zero in 20 days treatment. However, this result was not achieved in the groups treated with Akbar 1 and silver sulfadiazine ointment. These results also indicated the higher bactericidal feature of ZOUSH ointment compared to Akbar 1 and silver sulfadiazine ointments.In the group treated with ZOUSH ointment, during the treatment, the mean count of isolated P. aeruginosa 5 days after the treatment was higher than the mean count of isolated P. aeruginosa 10 days after the treatment. It can be due to the inability of the immune system to eliminate the bacterium. However, the mean count of isolated P. aeruginosa 10 days after the treatment was less than the mean count of isolated P. aeruginosa 15 and 20 days after the treatment, which could be due to the improvement of the immune system ability to control the infection.In the group treated with the base ointment as well as the untreated group, the mean count of isolated In a study by Hua-Liang Li et al., the effect of Crocodile oil burn ointment (COBO) was evaluated on Wistar rats with second-degree burns. The analgesic and anti-inflammatory effects of COBO ointment were studied . In survEphedra alata aqueous extract has the ability to heal wounds in animal models and investigated the effect of the E. alata aqueous extract ointment with polyethylene glycol ointment base versus placebo ointment. Phytochemical studies showed that E. alata aqueous extract contains flavonoids, alkaloids, phytosterols, phenolic compounds, volatile oils, and tannins. The E. alata aqueous extract ointment had the good effect on wound healing compared to placebo ointment. In this study the effects of ZOUSH ointment, carvacrol and thymol were the active ingredients of S. khuzestaniea, Z. multiflora, and O. vulgar, and the results showed wound healing in compared with the non-treated group and the group treated with ointment base [In a study by Pannerselvam et al., the effect of silver nanoparticle-impregnated cotton fabric was investigated on the healing rate of wound burns in Wistar rats versus commercial ointments-impregnated cotton fabrics. The results indicated a faster improvement in the group treated with silver nanoparticles-impregnated with cotton fabric compared to other groups. The treatment period was 18 days, which is close to the duration of treatment with ZOUSH ointment . Kittanaent base .P. aeruginosa PAO1, the inhibition zone diameter for ZOUSH ointment was reported 188 mm using the 60 μg/ml disk.To evaluation the effect of ZOUSH ointment, the antibacterial properties of ointment compositions was measured against Gene expression is a process resulting in the synthesis of a functional product using information stored in the gene. The gene expression is evaluated by several methods, such as Real-time PCR –59.exoS, lasA, lasB and gyrA genes in P. aeruginosa. Most of the studies have been conducted so far to investigate the antibacterial properties herbal compounds used MIC, disk diffusion, and RT-PCR methods.In this study, Real-time RT-PCR technique was used to study the effect of ZOUSH ointment compounds on inhibition the expression of S. khuzestaniea was investigated on the mexR and mexA genes in P. aeruginosa. The results showed that low expression of these genes was due to carvacrol compounds [S. khuzestaniea on reduction the expression of virulence genes P. aeruginosa [In a study by Jalalvand et al., the effect of ompounds . In a reruginosa .S. khuzestaniea on the expression of exoenzyme S, exotoxin A, secretory systems and efflux pump genes of P. aeruginosa was investigated by semi-quantitative RT-PCR technique [In a study by Esmaeili et al., the inhibitory effect of echnique .S. khuzestaniea on exotoxin A P. aeruginosa by semi-quantitative RT-PCR technique [Abbasi et al. identified the inhibitory effect of echnique .Jalali et al. employed the semi-quantitative RT-PCR technique to optimize the analysis of CPTI gene expression in trout fish liver .S. khuzestaniea and Z. multiflora essential oils on the control of aphA-6 gene expression in MDR Acinetobacter baumannii by Real Time-PCR technique. The results showed that carvacrol is the main compound of S. khuzestaniea and thymol is the major compound of Z. multiflora. The MIC was 0.3 μl/ml for S. khuzestaniea and 0.45 μl/ml for Z. multiflora. The aphA-6 gene encodes aminoglycoside transferase enzyme, thereby providing resistance [Saghi et al. investigated the effect of sistance .S. khuzestaniea alcoholic essential oil on the expression of the gene associated with Acinetobacter baumannii biofilm by the Real-Time PCR technique. The results showed that the presence of 90.88% carvacrol in the essential oil of S. khuzestaniea, and MIC was reported 0.3 μg/ml for S. khuzestaniea essential oil. In this study, the results of Real-time PCR were analyzed by SPSS software and ANOVA test. [Bahador et al. studied the effect of VA test. .exoS, lasA, lasB and gyrA genes in P. aeruginosa by Real-time RT-PCR technique.The present study was carried out to investigate the effect of herbal ZOUSH ointment on inhibition the expression of We can conclude that in a burn wound model in mice, ZOUSH ointment was able to decrease the healing process both biochemically and statistically as compared to SSD and the control group. Through its evident ant virulence gene effects, ZOUSH can be used as an alternative agent to wound healing therapies in the future. However, wound tissue healing is a multi-factorial process involving the groups of enzymes, receptors, gene silencing and healing. Therefore, further research is needed for the elucidation of the role of these processes on the rabbit and human model for the observed action of ZOUSH.


Kerwyn Casey (“KC”) Huang is an Associate Professor at Stanford University, studying the physical nature of biological systems and the underpinnings of fundamental processes such as cell shape determination, cell division, and intracellular and microbial community organization. In this interview, KC discusses how the ability to pursue insights at scales from molecules to cellular communities can shed new light on longstanding questions, the necessity for new tools in exploring the microbiome, how to create an empowering lab environment, and why integrating chemistry with physics and biology can bring us closer to asking the right questions. I have always been fascinated by simple, fundamental questions about how nature works. I focused on purely physical questions at first , but as a postdoc I accidentally wandered into biology and became hooked on a new set of unknowns. Many of the basic questions that my lab and I tackle now have been around for decades (if not centuries): how do cells break symmetry, how do they deal with starvation, how do they adjust to changes in temperature, pH, and osmolarity? It’s exciting to examine these biological questions with a multitude of new tools that allow us to explore scales all the way from single amino acids to communities of cells. If you had told me 10 years ago that I would be examining crystal structures in the morning and mouse tissue sections in the afternoon, I’d have said you were crazy. Now I can see that many seemingly different biological phenomena are actually related across space and time.Fig. 1. KC Huang. Website: http://whatislife.stanford.edu/The clichéd (but true) answer is that every project has provided inspiration, tough challenges, and memories—in part because so many of our projects ended up taking us places we could never have predicted. However, two projects that laid the foundation for our lab today come to mind.Escherchia coli cells not stop growing, they didn’t slow down at all [The first project was initialized by my former postdoc Rico Rojas (now an Assistant Professor at NYU), who was inspired by work in plants that suggested that turgor pressure (osmotic swelling) drives growth. Rico set out to determine how changes in turgor pressure affect the growth of bacteria. Almost a hundred years ago, Heinrich Walter discovered that bacterial colonies grow more slowly on agar plates at higher osmolarity, and one interpretation of this result is that the slowdown is due to the cells having lower turgor pressure. However, there are many other explanations involving transcriptional or structural adaptations. Rico used a microfluidic device to lower turgor pressure to zero almost instantaneously. To our surprise, not only did n at all . I learnThe second project emerged from conversations between myself and close friend and collaborator Justin Sonnenburg (Associate Professor of Microbiology & Immunology here at Stanford) as we prepared to teach a class on computational modeling of microbial communities. This class was as much an opportunity for the two of us to learn about each other’s expertise as it was to teach the students (this is the secret about great teaching: the instructors learn a lot too!). In talking with Justin about the gut microbiota, my mind naturally gravitated toward questions about the physical organization and dynamics of this organ. I was shocked by how little was known about these questions. This knowledge gap spurred our first collaboration, in which we developed computational and experimental tools to quantify the spatial organization of the mammalian gut. I was thrilled when our Teaching Assistants and the students in the course made important contributions to that project—again, this is the way that good teaching works. We made some really fun discoveries, such as the dramatic effects of dietary changes on the host mucosal lining and bacterial clustering . MoreoveI was trained as a physicist, so I encountered every paper that I read as I first started thinking about biology with a sense of wonder. This transition required me to reinvent myself as a scientist—a process that is ongoing even today. As a result, I especially admire and am inspired by other scientists who have undergone a renaissance. My postdoc advisor Ned Wingreen (Princeton University) made seminal contributions to condensed matter physics, and then transitioned to biophysics as a research scientist at NEC Research Laboratories. Ned has worked at the frontiers of a diverse range of fields including chemotaxis, self-organization, quorum sensing, liquid–liquid separations, and many others. Ned commits to identifying and answering the most important questions in any field, regardless of whether they are trendy at the time. I’ve sought to work this way myself, and to inspire my lab members to have the same intellectual courage.In the last several years, I’ve had the privilege of working with Carol Gross at UCSF. Carol has had an amazing career studying fundamental cellular components such as the heat shock response and RNA polymerase. And yet, around 10 years ago she completely shifted her lab to focus on the systems biology of bacterial stress responses. One of her first papers in this area is called the “Phenotypic Landscape of a Bacterial Cell”  and it’sLast but certainly not least, I’m inspired by all the students and postdocs in my lab who take on the scientific challenges that we pursue. Their willingness to embrace the great unknown—and to invent the ways to navigate it—is what has made our lab successful.Establishing a healthy work environment goes a long way toward having a happy, productive lab. For me, this means that all my lab members feel empowered to speak up when they have questions. We balance their scientific independence with a regular pattern of dialog between us. Above all, it’s important to me that my lab members are excited about their projects. Nearly all of my lab members are pursuing and publishing on multiple projects, and many of them collaborate closely with other labs as well. As a result, they are exposed to different ways of thinking about problems and a broad range of skills and tools, and they start forming their scientific network early on. In other words, I view my lab as a safe environment that encourages excellence in the skills that empower people to go on to be pioneers, deep thinkers, and good colleagues. My lab members frequently ask me how I deal with so many meetings each day, and I tell them that all those opportunities to discuss science is why I do this job.Learn chemistry. The more we think about how cells interact with their environment, the more I think about the omnipresent and ever-changing chemical milieu that envelops the cell. Someone recently told me that there are two types of scientists: people who study metabolism, and people who don’t know that they’re studying metabolism. I’m glad we’re finally in the first group, but I still have a long way to go to actually have intuition about how chemistry affects biology. My wife took a Biology major and a Chemistry minor in college, and when we were first dating, I saw a silver necklace on Etsy that was shaped as the chemical structure of a polypeptide. I showed it to her, and she read off the word “GEEK” as if it was written in English. I aspire to be that geeky. When you have a strong intuition about the way the universe works, you are more quickly attracted to the questions that really matter.My lab straddles many fields, but here I’ll answer for the microbiome field. Although microbiome research is built around cutting-edge -omics tools such as sequencing and mass spectrometry, there are so many areas in which new tool development would have a transformative impact. For example, there are tens of thousands of genes with novel function across the gut commensal bacteria, and yet our ability to perform genetic manipulation is currently limited to a few model organisms. Basic questions, such as which cells are growing when and where, remain unanswered because we lack tools to probe the gut spatially and dynamically. And even though sequencing can provide an amazing amount of insight, it is frustrating to have to wait for weeks or even months to examine data characterizing the gut community. With a turnaround of hours, long (multi-week) experiments could actually be adjusted on the fly.In the best cases, I believe that scientific publishing makes for better science: critical but fair reviewers help to improve the quality of the paper, and publication is viewed as an accomplishment to be celebrated by the authors and field . Having said that, most of us have worst-case experiences that are often independent of the science itself: the third reviewer having a bad day, or the “what to do” moment when another just-published paper has significant overlap with a paper you are about to send out for review. Some journals are now encouraging communication among reviewers, which helps to highlight consensus criticism rather than the outlier concerns. Some journals are adopting a policy in which almost-concurrent papers on the same topic are viewed as a welcome reproduction of science rather than a pre-empting of novelty. I’d like to see more of these sorts of community-themed innovations (I think of them more as “improvements”), which seem to arise from constructive dialog among editors, reviewers, and authors.Website:http://whatislife.stanford.edu/


Euglena gracilis transcripts of the tubA and gapC genes, which contain two types of introns: nonconventional and spliceosomal. The relative order of intron excision was compared for pairs of introns belonging to different types. Furthermore, intermediate products of splicing were analyzed using the PacBio Next Generation Sequencing system. The analysis led to the main conclusion that nonconventional introns are removed in a rapid way but later than spliceosomal introns. Moreover, the observed accumulation of transcripts with conventional introns removed and nonconventional present may suggest the existence of a time gap between the two types of splicing.Nuclear genes of euglenids and marine diplonemids harbor atypical, nonconventional introns which are not observed in the genomes of other eukaryotes. Nonconventional introns do not have the conserved borders characteristic for spliceosomal introns or the sequence complementary to U1 snRNA at the 5' end. They form a stable secondary structure bringing together both exon/intron junctions, nevertheless, this conformation does not resemble the form of self-splicing or tRNA introns. In the genes studied so far, frequent nonconventional introns insertions at new positions have been observed, whereas conventional introns have been either found at the conserved positions, or simply lost. In this work, we examined the order of intron removal from  Euglena gracilis genes. The relative order of intron excision was compared for pairs of introns belonging to different types. We also surveyed thousands of intermediate products of splicing using the Next-Generation Sequencing system. Summarizing the results of both experiments, we proved that spliceosomal introns are removed at an earlier stage of pre-mRNA maturation than nonconventional ones.The existence of conventional spliceosomal introns in genes of eukaryotic organisms is a well-known theorem. However, genes of the unicellular algae group, euglenids, contain also another type of introns, so-called nonconventional ones. They lack canonical borders, a feature most characteristic for conventional introns and form a stable secondary structure bringing together their ends. Along with the increasing popularity of whole genome studies, nonconventional introns were also disclosed in the genes of other protists, diplonemids. In this study we were particularly interested which introns–conventional or nonconventional–are removed earlier from euglenids’ pre-mRNA. To track this process we analyzed transcripts of the two  Nuclear genes of eukaryotes contain introns which are removed from pre-mRNA in a splicing process catalyzed by the spliceosome–a ribonucleoprotein complex assembled from five small nuclear RNAs (snRNA) and a range of proteins. Intron removal begins co-transcriptionally–when the pre-mRNA synthesis on the DNA template by RNA polymerase II is yet to be completed –5. This Trypanosoma cruzi), African trypanosomiasis (Trypanosoma brucei) or leishmaniasis (many species of Leishmania), (2) euglenids, including a large group of photosynthetic species with secondary plastids of green algal origin, (3) diplonemids, free-living phagotrophs, common predators dominating the marine environment and (4) poorly researched symbiontids, free-living flagellates inhabiting low-oxygen marine sediments [trans-splicing [Euglena gracilis (model euglenid species) genome sequencing project is in progress but available data are still far from satisfactory. The reason for that is the unexpectedly large size of the genome (about 2 Gbp) and high level of repetitive sequences. Only a few genes were annotated so far, but the presence of two types of introns was confirmed on the genomic level [The rules mentioned above describe the maturation of regular eukaryotic pre-mRNA containing merely typical, conventional, spliceosomal introns. However, this is not the only intron type to be found in eukaryotic protein-coding genes. Nuclear genes of some euglenozoans (supergroup Excavata) harbor atypical, nonconventional introns which are not observed in the genomes of other eukaryotes. The Euglenozoa group comprises: (1) kinetoplastids, including parasites causing diseases such as the Chagas disease , solely conventional (e.g. tubG), or introns of both types  occur. It is also worth mentioning that some nuclear transcripts of euglenids undergo trans-splicing, in which the splice-leader sequence deriving from the SL-RNA is added to the 5' end of a transcript in place of an outron, a semi-intron containing the branch point A, polypyrimidine tract and the 3' splice site AG [trans-spliced outron and the first cis intron were performed only for genes harboring exclusively either nonconventional introns (rbcS) or conventional ones (tubG). According to previous reports, in the rbcS gene, nonconventional cis-splicing occurs earlier than conventional trans-splicing [tubG gene, conventional trans-splicing takes place before conventional cis-splicing [tubA and gapC). The relative order of intron removal was compared for pairs of introns belonging to different types. Additionally, intermediate products of splicing were analyzed using the Next Generation Sequencing (NGS) technology of PacBio SMRT system (Pacific Bioscience) generating long NGS reads (on average up to 15–20 kbp long). Interestingly, the results obtained differed from previous findings.There are nuclear genes of euglenids in which exclusively nonconventional introns . This result proves that conventional trans-splicing may occur before nonconventional cis-splicing. Furthermore, the outcome is consistent with the results obtained for the tubA and gapC transcripts.Results obtained for onal one . TherefotubA and gapC transcripts, as well as to define the general order of splicing, single molecule real time sequencing of the three different PCR amplicons was performed: (1) amplicon obtained in reaction with primers complementary to the first exon and the last, fourth intron  in the gapC gene, using the cDNA from the reverse transcription reaction, with the primer complementary to the last, fourth intron as template, , using the cDNA from reverse transcription reaction with the primer complementary to the sixth, last intron as template , using the cDNA from the reverse transcription reaction with the primer complementary to the fifth intron as template , as well as for i6-  and i5-  tubA were successfully assigned to particular categories based on the alignments with the appropriate reference sequences.As a result of sequencing, raw PacBio reads of various lengths corresponding to three abovementioned amplicons were received. In total, 29,593 reads were obtained for the gapC, i6- and i5-tubA amplicons. After mapping according to the reference sequences and manual examination of contigs, about 4,049 unreliable reads for those three amplicons were discarded (in sum). Thus, 25,544 credible sequencing reads, fulfilling the criteria of complete amplification length were selected for further analysis. All of the accepted reads for The gapC amplicon produced three possible categories of sequencing reads, with: (I) the first conventional intron absent, (II) all introns present, and (III) all analyzed introns spliced out. Reads representing the first type were the most abundant and accounted for 75.49%  of the total pool of accepted sequences. The second category was represented by 24.40%  of reads, whilst only 0.11% (17) of reads was assigned to the third–the least abundant class. Among 14,960 analyzed sequences, reads with the conventional intron present, but with the nonconventional introns absent, were not found .tubA gene (i6-tubA amplicon), 11 categories of splicing intermediates were distinguished  were divided into four categories  is preferentially removed as first , while for the i6-tubA one, 2,782 . Though it is worth mentioning that the methodology adopted in the experiment was based on the amplification procedure, implying that the number of obtained reads does not necessarily directly reflect the number of intermediates present in the cell. Nevertheless such a picture may indicate that these types of transcripts accumulate before the dynamic nonconventional splicing begins. This, in turn, may suggest existence of some factor delaying the initiation of nonconventional intron removal. It cannot be ruled out that the different localization of both types of splicing may play a role here. Conventional splicing is tightly coupled with transcription–it starts before a pre-mRNA molecule synthesis completion. Perhaps nonconventional splicing cannot launch at such a stage (maybe due to the lack of splicing factors?) and constitutes a post-transcriptional process, which begins only when the released pre-mRNA molecules, nonetheless lacking conventional introns, reach the appropriate localization in the nucleus. Moreover, it should not be ruled out that nonconventional splicing may take place much later, even in the cytoplasm. However, in order to confirm this hypothesis, more insightful research would have to be done in the future. Unfortunately, due to the lack of a standardized method of investigating transcripts’ localization in E. gracilis. This constitutes another portion of knowledge about atypical nonconventional introns, a research area still extremely challenging due to the lack of well-established methods. Further studies will be needed to answer some of the remaining questions. What is the mechanism of atypical nonconventional intron removal? Is the theory of their transposon origin correct? Likewise, how may their propagation in nuclear genomes be related to the activity of DNA repair systems (as it was hypothesized previously) [In this article, we summarize a series of experiments determining the order of processing of conventional and nonconventional introns, carried out for viously) . AnswersEuglena gracilis strain Z (SAG 1224-5/25) used in this study was originally provided by Culture Collection of Algae at Göttingen University , Germany and maintained at a repository of the Department of Molecular Phylogenetics and Evolution at the University of Warsaw, Poland. Cells were cultured in the Cramer-Myers medium [-2 s-1 provided by cool white fluorescent tubes). Circa 1 × 107 cells in the logarithmic growth phase were harvested by centrifugation , and washed twice with 5 ml of nuclease-free water to completely remove the medium residues.The s medium , supplemTotal cellular RNA was extracted immediately from freshly collected cells with the mirVana miRNA Isolation Kit (Invitrogen) according to the manufacturer’s protocol designed for total RNA isolation. The RNA sample was treated with DNase in solution with the DNAfree DNA Removal Kit (Invitrogen) between the organic extraction and transfer onto a glass fiber filter. This procedure was performed according to the rigorous digestion conditions described in the manufacturer's instructions.The purity and concentration of the isolated RNA was examined spectrophotometrically using NanoPhotometer NP80 (Implen) and fluorometrically using Qubit 3.0 (Life Technologies). The integrity of the RNA sample was also checked via electrophoresis under denaturing conditions  and run with a 1x TT buffer , as described in detail by .rbcS transcript  [tubA and gapC genes , 0.2 mM dNTPs, 1x reaction buffer (Invitrogen) and 200 U of SuperScript III Reverse Transcriptase (Invitrogen). 10 pmoles gene specific reverse transcription primers were used in cDNA synthesis in order to (1) reinvestigate the maturation of the 5’ end of the ctively)  and to (Products of cDNA synthesis were used as a template in PCR reactions carried out as follows. A standard 25 μl reaction mixture contained 0.5 U of Phusion High-Fidelity DNA Polymerase (Thermo Scientific), 0.2 mM dNTPs, 10 pmol of each primer, 1x reaction buffer HF (Thermo Scientific), 1.2 μg of Taq Single-Stranded DNA Binding Protein (EURx) and 1 μl of the first strand cDNA or 1 μl of the negative control reaction from the previous step. 36 cycles of PCR were performed. After an initial 30 s at 98°C, each cycle consisted of 10 s denaturation at 98°C, 15 s annealing at a temperature optimal for each primer set  and primgapC, tubA and rbcS genes–as shown in E. gracilis, retrieved from the GenBank database .Amplified DNA fragments intended for NGS  were purified directly from the solution with the GeneMATRIX PCR/DNA Clean-Up Purification Kit (EURx), according to the manufacturer’s instructions. PCR products intended for Sanger sequencing  instrument. Sequencing reads were initially quality controlled and trimmed by the sequencing institution. In addition, a FastQC 0.11.7 [gapC and tubA genes respectively were saved in separate FASTQ files and converted to FASTA format using the Geneious Basic 10.2.2 software [gapC and tubA contigs was mapped using Geneious mapper algorithm against the reference sequences of gapC (GenBank accession number: L39772.1) and tubA (GenBank accession number: AF182557.1) genes of E. gracilis, respectively. The following mapping parameters were applied: medium-low sensitivity/fast mapping with multiple best matches set to all, finding short insertions and large deletions up to 1000 bp as well as including insertions in structural variants, minimum support for structural variant discovery = 2; without trimming. Only successfully mapped, complete reads–flanked by PCR primers at both ends (which indicates complete PCR product amplification) were taken into consideration in the further analysis. Incomplete reads (without the sequence(s) of primers), as well as reads extending beyond the primer binding site(s) were discarded. Reads containing ambiguous nucleotides  close to and/or located within the intron/exon junctions were also excluded, as being equivalent to potentially defective products of intron excision. Remaining fair reads were subsequently compared and categorized based on the presence or absence of individual intron(s) in the obtained alignment. Mapped sequences were then sorted based on the alignment length. Afterwards, all possible read types for both studied genes were annotated and counted. The most probable order of intron removal has been inferred from the summarized statistics of the reads.The amplicons obtained as a result of RT-PCR reactions for the C 0.11.7  analysissoftware . All reaS1 TablePrimers used to generate amplicons are marked with an asterisk.(PDF)Click here for additional data file.S1 FigtubA (A) and gapC (C) genes, respectively. Schematic depiction of products obtained with each primer set for both studied genes . Arrows represent primers, lines represent PCR products. Gaps within spliced products  refer to the absent intronic sequences. Product lengths are given alongside depictions.PCR primers used for the pairwise comparison of removal of adjacent introns in (PDF)Click here for additional data file.


Therefore, the identification of biomarkers that differentiate these diseases is of importance for preventing misdiagnosis. In this regard, the present study aimed to identify the disorders at the early stages, based on lung transcriptomics data and drug-target interactions.To this end, the differentially expressed genes were found in each disease. Then, WGCNA was utilized to find specific and consensus gene modules among the three diseases. Finally, the disease-disease similarity was analyzed, followed by determining candidate drug-target interactions.The results confirmed that the asthma lung transcriptome was more similar to COPD than IPF. In addition, the biomarkers were found in each disease and thus were proposed for further clinical validations. These genes included RBM42, STX5, and TRIM41 in asthma, CYP27A1, GM2A, LGALS9, SPI1, and NLRC4 in COPD, ATF3, PPP1R15A, ZFP36, SOCS3, NAMPT, and GADD45B in IPF, LRRC48 and CETN2 in asthma-COPD, COL15A1, GIMAP6, and JAM2 in asthma-IPF and LMO7, TSPAN13, LAMA3, and ANXA3 in COPD-IPF. Finally, analyzing drug-target networks suggested anti-inflammatory candidate drugs for treating the above mentioned diseases.In general, the results revealed the unique and common biomarkers among three chronic lung diseases. Eventually, some drugs were suggested for treatment purposes. Lungs are considered as vital and vulnerable parts of the respiratory system and play critical roles in the body  are struggling with COPD. The incidence rate has indicated an increase of 44.2% from 1990 to 2015  were estimated based on healthy and patient states for the samples . The mean of expression was used for multiple probes mapping to the same gene  . The gene list of shared DEGs between the two diseases (three states) and among the three diseases was used for further investigations Fig. .Fig. 1WAccording to the study by Horvath and Dong  was calculated using the pickSoftThreshold function. Then, disease-specific adjacency matrices were generated utilizing the selected β threshold and were transformed to the Topological Overlap Matrix (TOM) using the TOMsimilarity function : The correlation between gene expression profile in the modules and the disease states, as well as the correlation between the MEs and the gene expression were referred to as Gene Significance (GS) and Module Membership (MM), respectively. Modules with a P < 0.05 were filtered in the MM-GS analysis for each disease.Analysis of determining the genes associated with the disease: The DisGeNET (version 5.0) online database was used to determine the number of genes associated with each disease within the significant modules that were obtained in previous steps (a and b)  is defined as the first principal component of the gene expression in each module. In the present study, the MEs were combined using the mergeCloseModules function based on their similarity in the profile of the gene expression. This function uses a cutHeight parameter to determine the correlation among the MEs. The parameter was set to 0.15 in this study, and modules with a correlation ≥0.85 (1-cutHeight) were merged accordingly. Finally, three subsequent analyses were used to identify the most important modules for each disease as follows   and pathway databases, including the Kyoto Encyclopedia of Genes and Genomes (KEGG), Biocarta, and Reactome via the Enrichr  was utilized to identify the candidate drugs in the studied lung diseases  and GSE23611 (GPL6480) were used in this study. After data normalization and quality control, 34 COPD samples  and 32 healthy controls, as well as 118 IPF samples and 86 healthy controls from the GSE47460 dataset were utilized to determine the DEGs. In addition, 26 samples from asthmatic patients and 7 healthy controls were selected from GSE23611. The results of differential analyses are displayed in Fig. The DEGs with a matching up/down-regulated pattern between two or among three diseases were nominated as the DEGs with Identical Pattern (DIPs) to assess disease-disease expression similarity. The results of the three pairwise comparisons revealed 616, 615, 1452, and 304 shared DEGs between asthma-COPD, asthma-IPF, COPD-IPF, and asthma-COPD-IPF, respectively. Table Based on the results, there are more DIPs between asthma and COPD compared to COPD and IPF, as well as asthma and IPF. However, there are more DIPs between COPD and IPF compared to asthma and COPD or asthma and IPF.Disease-specific DEGs  were used to construct three disease-specific co-expression networks. The optimal β parameter for obtaining a scale-free network was computed as 11, 15, and 21 for COPD, IPF, and asthma, respectively  were constructed, including asthma-COPD, COPD-IPF, and asthma-IPF. Each CN was used to extract the Consensus Modules (CMs). The plots of β parameter computation for the constructed CNs are provided in Additional file A CN including 304 shared DEGs was reconstructed to extract the CMs among the three diseases. However, no CM could be extracted using the WGCNA.Enrichment analysis was performed to functionally evaluate the gene sets that were involved in specific modules, CMs, and overlapping DEGs among three diseases as follows.Table Some of the enriched significant biological pathways and GO biological processes of the CMs are reported in Table Table The candidate drug-target interactions were investigated after identifying consensus and specific modules among three diseases. Figure Figure Figure Progressive chronic respiratory diseases are associated with a persistent and progressive decline in lung function and exacerbations, which require smart diagnosis and treatment. Decreasing the rate of progression, staging, and exacerbations in chronic respiratory diseases such as asthma, COPD, and IPF is critically vital in controlling mortality -beta signaling pathways. These pathways are involved in the inflammation of the respiratory tract, immune system, and chronic inflammatory diseases  were investigated after evaluating the disease-specific modules. Also, two modules (Turquoise and Pink) for asthma-COPD, one module (Purple) for asthma-IPF, and three modules  for COPD-IPF pairs were identified after the module-trait analysis. Biological pathways and GO analysis conducted for the genes, are involved in each significant consensus module.Based on the results of the biological process analysis for the asthma-COPD, the genes of the turquoise module were mainly enriched in cilium assembly, cilium organization, cilium movement, and plasma membrane-bounded cell projection assembly. These processes were due to the dysfunction of the bronchial epithelial and the disruption of cilia function as the important features of asthma and COPD diseases  were identified in asthma-IPF among ten hub DEGs. In the purple module, COL15A1 is involved in the smooth muscle cell (SMC) phenotype that is controlled by the epigenetic state, so that the hypomethylation of COL15A1 transpires during SMC proliferation and the consequent increased gene expression may affect the SMC phenotype (Connelly et al. Ca2+-dependent manner. It also plays a vital role as a mediator in cellular growth, signal transduction pathways, cancer proliferation, invasion, and migration (Wang et al. The results of the biological process analysis for COPD-IPF represented that the genes of the blue module are enriched in immune processes, inflammation, and remodeling. Nine novel hub DEGs were specified in COPD-IPF, including GPM6A, ARHGEF26, ANKRD29, LMO7, TSPAN13, LAMA3, ST6GALNAC5, ANXA3, and CLIC5. In the blue module, LMO7 is known as a negative feedback regulator of TGF-β signaling and extracellular matrix deposition (Xie et al. Previous studies have only evaluated the overlap mechanism between two diseases whereas the present study considered three diseases together and could identify the focal biological processes for all three diseases. These results were not observed due to the presence of other genes available in the top rank between two diseases, and the statistical analysis led to these processes that were not shared in the overlap of two diseases. Likewise, the processes observed in COPD and IPF confirmed the irreversibility of these diseases whereas a cure is suggested in asthma.Based on the data in Tables Drug-target identification for consensus and common DEGs in three diseases revealed that based on specific DEGs, tretinoin as a retinoid drug is recommended for all three diseases. Retinoids are involved in many essential functions of the body such as the immune system, cell proliferation, and differentiation, bone growth, the activation of tumor suppressor genes, as well as natural and malignant epithelial cell growth in various tissues (Buckley et al. Bevacizumab is one of the VEGF inhibitors that can be identified as a candidate for COPD (Papaioannou et al. Low-dose paclitaxel is one of the TGF-β1/Smad3 pathways suppressing drugs, and in this study, it was identified as a candidate drug for IPF. TGF-β1 is responsible for creating a cascade of incidents leading to fibrosis, and the abnormal activation of TGF-β1 plays a vital role in the pathogenesis of IPF (Wang et al. Likewise, sirolimus as an immunosuppressant drug is suggested as a candidate for the asthma-COPD therapy. This drug inhibits the activation of T and B-cells by inhibiting mTOR and plays an essential role in the biogenesis of the ribosome, cell cycle progression, lipid synthesis, mitochondrial biogenesis, and autophagy of COPD (Miłkowska-Dymanowska et al. Tacrolimus is one of the candidate medicines in asthma-IPF as well. This drug inhibits the transcription of IL-2 and several other cytokines, especially in T-helper lymphocytes (Horita et al. In addition, cyclophosphamide is regarded as one of the candidate drugs for COPD-IPF. The combination of this drug with corticosteroids is used to reduce the severity of IPF (Emura and Usuda The current study proposed unique and common biomarkers and drugs among the three diseases. Moreover, it was confirmed that asthma lung transcriptome is more similar to COPD than IPF. These findings could help the researchers to differentiate the mentioned diseases. However, this study has some limitations including ignoring the side effects of the drugs. Additionally, novel biomarkers were proposed computationally. Therefore, the clinical studies are recommended to confirm the validity and reliability of the results of this study, as well as those of similar studies in the future.The findings indicated that the proposed biomarkers in chronic lung diseases might serve as predictive targets among the three diseases. They include RBM42, STX5, and TRIM41 in asthma, CYP27A1, GM2A, LGALS9, SPI1, and NLRC4 in COPD, as well as ATF3, PPP1R15A, ZFP36, SOCS3, NAMPT, and GADD45B in IPF. In addition, other identified genes were LRRC48 and CETN2 in asthma-COPD, COL15A1, GIMAP6, and JAM2 in asthma-IPF, along with LMO7, TSPAN13, LAMA3, and ANXA3 in COPD-IPF. Finally, anti-inflammatory drugs such as corticosteroids were proposed for treating the three diseases.Additional file 1. The list of DIPs and shared DEGs among three diseases.Additional file 2 Selecting the optimal β parameter for disease-specific networks.Additional file 3. The list of all gene symbols in candidate disease-specific modules.Additional file 4. The list of selected modules for three diseases.Additional file 5 Selecting the optimal β parameter for consensus networks.Additional file 6. The list of all gene symbols in candidate consensus modules.Additional file 7. Significant biological pathways and GO Biological Processes in disease-specific modules.Additional file 8. Significant biological pathways and GO Biological Processes in consensus modules.Additional file 9. Significant GO biological processes of overlapping DEGs among three diseases.Additional file 10:. The list of candidate disease-specific drug-target interactions.Additional file 11. The list of candidate consensus drug-target interactions.Additional file 12. The list of candidates shared drug-target interactions among three diseases.


The WSx) electrocatalyst for hydrogen evolution reaction (HER), researchers are pursuing inexpensive and highly efficient materials as an electrocatalyst for large scale practical application. Layered transition metal dichalcogenides (TMDCs) are promising candidates for durable HER catalysts due to their cost-effective, highly active edges and Earth-abundant elements to replace Pt electrocatalysts. Herein, we design an active, stable earth-abundant TMDCs based catalyst, WS Davodi and co-workers [2O3/MWCNTs functionalized with nitrogen-rich emeraldine salt for alkaline HER and OER processes. Luo et al. also demonstrated Fe3O4@NiFexOy core−shell nano-heterostructures toward the OER with an overpotential −410 mV@1 mA cm−2 and Tafel slope of 48 mV dec−1 [2/graphene/Ni foam as a catalyst in HER application and observed the low overpotential of −119 mV@10 mA cm−2, the small Tafel slope of ~43 mV dec−1, and the large cathodic current density. Recently, Zhou et al. has reported ternary tungsten sulfoselenide (WSx)2(1−Sex2) particles with a 3D porous metallic NiSe2 foam to have excellent catalytic performance with −88 mV@10 mA cm−2 of overpotential, 46.7 mV dec−1 of Tafel slope, and 214.7 μA cm−2 of exchange current density [2 QDs film using a solution process and WS2/CoSe2 heterostructure for HER applications [x)2(1− Se2x and WSx)2(1−Sex/graphene/NF (51 mV dec−1) as compared to the WS2/NF and WS2/graphene/NF .Recently, many efficient strategies to increase the number of active edge sites with large surface areas, high porosity, and better intrinsic electrical conductivity or the contact between the catalyst and the electrode were adopted to increase the electrocatalytic activity of electrode material. Herein, we utilized a 3D porous structure nickel (Ni) foam (NF) as a highly conductive skeleton, and produced WS2)/argon (Ar) (30/50 sccm) environment at 900 °C for 30 min in a quartz tube furnace to clean the surface of the Ni foam without breaking the vacuum. Then to prepare the graphene on NF, the mixture of H2/Ar/methane (CH4) flow (H2/Ar/CH4 = 50:100:50 sccm) was maintained for 30 min. Subsequently, the H2/CH4 flow gas channel was shut off, and then rapidly cooled to room temperature in Ar environment. Furthermore, prepared graphene/NF was used as substrate for the growth of WS2 and WSx) was cleaned with ultrasonic baths of acetone, ethanol, and deionized water, baked at 120 °C for 5 min, and then annealed with a hydrogen 2WS4)  was used as the main source material. First, a precursor of (NH4)2WS4 (0.2 g) was dissolved in N,N-Dimethylformamide (DMF) (20 mL), and then the solution was sonicated for 30 min. The 3D graphene/Ni foam was immersed into the prepared (NH4)2WS4 solution and then baked at 100 °C for 30 min. Finally, synthesized films were placed in an annealing chamber and heated up to 450 °C for 30 and 45 min in a sulfur or sulfur/selenium environment to form WS2/Graphene/NF and WSx)(1−Sex/Graphene/NF. The gas flow Ar/H2 flux (50/50 sccm) was maintained, and the pressure of chamber was kept at 2 × 10−2 Torr. The same quantity of sulfur/selenium (0.3/0.3 g) powder was used.For WS−1 in 0.5 M H2SO4 electrolyte at room temperature. For the LSV measurement, a saturated calomel reference electrode (SCE) was used as the reference electrode. WS2(30 min)/NF, WS2(45 min)/NF, WS2/graphene/NF, and WSx) scale with the help of the following equation: E(RHE) = E(SCE) + E°(SCE) + 0.059 pH. Electrochemical impedance spectroscopy (EIS) measurements were carried out in a potentiostatic mode with a frequency range from 0.01 Hz to 100 kHz under an amplitude of 10 mV. All the LSV polarizations were recorded after the ohmic drop iR correction. The stability measurement was examined using a chronoamperometric analysis.The electrochemical measurements were conducted in a three-electrode setup with a Biologic SP-300 workstation. The polarization curves were collected using a linear sweep voltammetry (LSV) with a scan rate of 10 mV.s2 was further grown by a hydrothermal process using an (NH4)2WS4 precursor on 3D graphene with NF. To improve the crystalline quality, the film was further annealed at 500 °C in a sulfur environment at 30~45 min to form WS2 nanoparticle-decorated graphene on NF (WS2/graphene/NF). To form WSx)(1−Sex nanoparticles-decorated graphene on NF (WSx)(1−Sex/graphene/NF), a CVD deposited film was annealed at 450 °C in a sulfur and selenium environment at 30 min, respectively, to form WS(1−x)Sex onto graphene. The schematic representation is given in Initially, the graphene was grown on a 3D NF using a chemical vapor deposition (CVD), as reported previously . WS2 was2/NF, WS2/graphene/NF, and WSx) as presented in x). Ni signals are ascribed from the NF substrate. The elemental mapping images of WS2 and WSx)(1−Sex layers are provided in x) and a high-resolution transmission electron microscopy (HRTEM).  bare NF a is comp bare NF b, the ree nature c. The naM images d. In thex)(1−Sex/graphene/NF particles as reported in previous literature [x)(1−Sex/graphene/NF. From the spectrum of graphene/NF (−1), 2D band (2706 cm−1), and D band (1364 cm−1) were exhibited due to defects in the carbon lattice [2/NF, two prominent Raman peaks originated at 350.1 and 420.5 cm−1 correspond to the E12g and A1g modes, respectively [2/graphene/NF, E12g, and A1g modes (351.6 and 420.5 cm−1) for WS2, two sharp peaks for graphene (G band: 1577.8 cm−1 and 2D band: 2699.8 cm−1) are shown [12g mode peak was observed at 250.6 cm−1, corresponding to WSe2 [x)(1−Sex/graphene/NF were further investigated via an X-ray diffraction (XRD) and X-ray photoelectron (XPS), respectively. The XRD patterns of graphene, WS2/NF, WS2/3D graphene, and WSx)(1−Sex/graphene/NF samples are shown in 2/NF (2 (WS2: JCPDS 657515). For WS2/graphene/NF, (004), (100), (101), (102), and (105) peaks were observed for WS2. In the case of WSx), (004), (104), (105), (112), and (200) lattice planes appeared in addition to the WS2/graphene/NF peaks as shown in 2: JCPDS No. 89-5257). From the XPS survey scan of WS2/graphene/NF (x)(1−Sex/graphene/NF (7/2 and W 4f5/2 (36.4 and 34.5 eV) doublets, which were indicative of the oxidation state of W4+, appeared. The S 2p1/2 and 2p3/2 orbital peaks observed at 163.6 and 161.9 eV, respectively, indicating the S2, confirmed the WS2 crystal [5/2 (53.8 eV) and Se 3d3/2 (55.6 eV) corresponding to the −2 oxidation state of selenium [The structural and chemical composition of the graphene, WS in 2/NF b, the pephene/NF , the obsphene/NF a, an add crystal ,35. The −1 in 0.5 M sulfuric acid (H2SO4) electrolyte solution by linear sweep voltammetry (LSV) with iR correction. As expected, the commercial Pt wire exhibited the lowest overpotential, which was close to zero. The WSx) for a geometric current density of 10 mA cm−2. In contrast, WS2(45 min)/NF, and WS2/graphene/NF exhibited inferior HER activity  (2 adsorption onto the Se facets than the S facets [HER activities were investigated via a standard three-electrode setup with a scan rate of 10 mV sctively) a,b. EarlS facets . Graphenx) hybrid catalyst (−97.2 mV@ 10 mA cm−2) [2/reduced graphene oxide hybrid nanosheets (−150 ~ −200 mV@10 mA cm−2) [2-WS2 (−129 mV@10 mA cm−2) [2@hollow nitrogen-doped carbon nanofibers (−185 mV@10 mA cm−2) [2 2D-nanohybrid electrocatalysts (−140 mV@10 mA cm−2) [x)(1−WxS2 hollow nanospheres on an Ni3S2 nanorod (−98 mV@10 mA cm−2) [2  on NF were used as electrocatalysts, and their LSV curves are provided in the The catalytic overpotential (−93 mV) of the WSmA cm−2) , WS2/redmA cm−2) , MoS2-WSmA cm−2) , WS2@holmA cm−2) , graphdimA cm−2) , and Mo(mA cm−2) . For com−1 was extracted for WSx)/graphene/NF and WS2/NF, respectively (−1 of WSx) [2/CC (127~105 mV dec−1) [2 and WS2 nanosheets on bare oxidized carbon fiber (OCF) (149 ~ 99 mV dec−1) [2 @WS2 nanorattles, WS2 nanoflakes and bulk WS2  [2 nanosheet/graphene (73 mV dec−1) [2 and WSx)2(1−Sex2 nanotubes (105 and 99 mV dec−1) [2 and WSx)2(1−Sex2 with a tunable band gap (100 and 85 mV dec−1) [The inherent property of catalytic activity for HER kinetics was probed by extracting the slopes from the linear regions in Tafel plots. A Tafel slope of 51 mV decectively c. The Tad for WS2 min/grapV dec−1) , bulk WSV dec−1) , WS2 @WSV dec−1) , verticaV dec−1) , and monV dec−1) .j0) was found to be ~0.162, ~0.165, and ~0.274 mA cm−2, for WS2/NF, WS2/graphene/NF and WSx)Sex/graphene/NF may be attributed to the large number of exposed active edge sites, good electrical conductivity, or the porous structure. The observed HER parameters for different electrodes are listed in −1, or a combination reaction ) that leads to a Tafel slope of 30 mV dec−1.The exchange current density (−1 for Pt exposes the hydrogen production proceeds with the fast discharge step (Equation (1)) followed by the Tafel (Equation (3)) [−1  suggest that hydrogen production proceeds with the fast discharge step (equation 1) followed by the Tafel (Equation (3)) or Heyrovsky ion-atom reaction (Equation (2)) [x)) ,54. The ion (2)) ,55,56. TRct) of Pt, WS2(45 min)/NF, WS2/graphene/NF, and WSx)/NF, WS2/graphene/NF, and WSx)(1−Sex/graphene/NF catalyst showed good HER catalytic properties in acidic electrolyte with an overpotential of −93 mV to drive 10 mA cm−2, a small Tafel slope of 51 mV dec−1, and a high exchange current density with excellent long-term durability. Our results proved that Se incorporated WS2/graphene/NF exhibits the highest electrocatalytic activity for HER, and it is stable in acidic media over a long period among the other electrodes due to high active edge sites and porous structures.In summary, an effective and efficient strategy was adopted for the synthesis of WS


New Specific Application Domain (SAD) heuristics or design principles are being developed to guide the design and evaluation of mobile applications in a bid to improve on the usability of these applications. This is because the existing heuristics are rather generic and are often unable to reveal a large number of mobile usability issues related to mobile specific interfaces and characteristics. Mobile Electronic Data Capturing Forms (MEDCFs) are one of such applications that are being used to collect health data particularly in hard to reach areas, but with a number of usability challenges especially when used in rural areas by semi literate users. Existing SAD design principles are often not used to evaluate mobile forms because their focus on features specific to data capture is minimal. In addition, some of these lists are extremely long rendering them difficult to use during the design and development of the mobile forms. The main aim of this study therefore was to generate a usability evaluation checklist that can be used to design and evaluate Mobile Electronic Data Capturing Forms in a bid to improve their usability. We also sought to compare the novice and expert developers’ views regarding usability criteria.included utility, clarity, question naming, categorization and measurability, with utility and measurability having a higher weight respectively. We then determined the proportion of participants who agreed (scored 4 or 5), disagreed (scored 1 or 2) and were neutral (scored 3) to a given criteria regarding a particular question for each of the experts and novice developers. Finally, we selected questions that had an average of 85% agreement (scored 4 or 5) across all the 5 criteria by both novice and expert developers. ‘Agreement’ stands for capturing the same views or sentiments about the perceived likeness of an evaluation question.We conducted a literature review in August 2016 using key words on articles and gray literature, and those with a focus on heuristics for mobile applications, user interface designs of mobile devices and web forms were eligible for review. The data bases included the ACM digital library, IEEE-Xplore and Google scholar. We had a total of 242 papers after removing duplicates and a total of 10 articles which met the criteria were finally reviewed. This review resulted in an initial usability evaluation checklist consisting of 125 questions that could be adopted for designing MEDCFs. The questions that handled the five main categories in data capture namely; form content, form layout, input type, error handling and form submission were considered. A validation study was conducted with both novice and expert developers using a validation tool in a bid to refine the checklist which was based on 5 criteria. The criteria for the validation The validation study reduced the initial 125 usability evaluation questions to 30 evaluation questions with the form layout category having the majority questions. Results from the validation showed higher levels of affirmativeness from the expert developers compared to those of the novice developers across the different criteria; however the general trend of agreement on relevance of usability questions was similar across all the criteria for the developers. The evaluation questions that were being validated were found to be useful, clear, properly named and categorized, however the measurability of the questions was found not to be satisfactory by both sets of developers. The developers attached great importance to the use of appropriate language and to the visibility of the help function, but in addition expert developers felt that indication of mandatory and optional fields coupled with the use of device information like the Global Positioning System (GPS) was equally important. And for both sets of developers, utility had the highest scores while measurability scored least.The generated checklist indicated the design features the software developers found necessary to improve the usability of mobile electronic data collection tools. In the future, we thus propose to test the effectiveness of the measure for suitability and performance based on this generated checklist, and test it on the end users (data collectors) with a purpose of picking their design requirements. Continuous testing with the end users will help refine the checklist to include only that which is most important in improving the data collectors’ experience. Over the years, electronic data collection systems are increasingly being used in health care particularly for data collection and management in health surveys, surveillance and patient monitoring . ElectroUsability is considered as one of the top attributes of assessing quality and its major role is to ensure that the interfaces are easy to use and that users are supported in performing their tasks efficiently and effectively . One of Heuristics that are applicable in one context may not work in another, or may sometimes contradict a heuristic used in another context. Secondly, their being broad often leaves room for the individual evaluator’s interpretation of what they mean, and may also be challenging to less experienced evaluators in finding pertinent design problems , 10. TheThe study therefore sought to generate and validate a design checklist for MEDCFs. We proposed a list of sub-heuristics from literature which focus on the data capturing process. We later validated this list in order to transform and refine it, so that it would be more usable to the intended users.To develop a design checklist for mobile electronic data collection forms (MEDCFs), we used different types of techniques and involved different stakeholders. First we conducted a literature review where we generated an initial usability evaluation checklist. The initial checklist was then validated by a team of software developers using a validation instrument.usability’, ‘evaluation’, ‘mobile’ and ‘heuristic’ and were left with 225 articles. We screened the abstracts and removed those papers which were not in English and those which were not about usability evaluation of user interfaces, leaving us with 134 articles. We then omitted those papers which were not focused on generating or validating usability heuristics for mobile devices or interfaces, and we were then left with 10 articles.We used the ACM digital library, Springer, IEEE-Xplore and Google scholar, together with some literature on best practices from other sources like Google. Our key terms in the search included ‘usability’, ‘usability evaluation’, ‘heuristics’, ‘mobile’, ‘tool’, ‘checklist’, ‘user interface’ and ‘design’. The key words were chosen with a focus on improving usability in mobile electronic data collection based on the assumption that usability can be improved through proper designing and evaluation of the user interfaces by developers using heuristics/design principles that are specific to MEDCFs. The inclusion criteria included papers that were focused on computer applications, usability, heuristic evaluation, generation and validation of heuristics.. Our search generated 242 papers for review after removing the duplicates. On screening based on titles, we then removed 17 papers whose titles did not have the words ‘We derived our usability evaluation questions from 10 papers , the majority of which came from a system checklist by Pierotti , 16.form content, form layout, input process, error handling and form submission [visual feedback and list pickers among others. The 125 questions were categorized as follows. Form content had a total of 35 questions, form layout had a total of 43 questions and the input process category comprised of 22 questions. Error handling had 23 questions, while form submission had the least with only 2 questions.We selected those sub-heuristics that fitted in the 5 categories and are representative of the data capturing process as shown in the design of web forms. This included transfer of sub heuristics from their original sub-heuristic category and placed under a new one based on what they are evaluating. The categories comprised of the bmission . We thenbmission , and yetThe form content depends on the data being collected. Some forms may be in form of questionnaires, whereas others may be in tabular form, hence the variation in content. The main content is usually questions and labels or fields of text entry. However, it is very crucial to map the environment which the users are familiar with in order to ease the use of the form. In this case, designing an electronic form that is analogous to the paper forms will quicken the data collectors’ understanding of the form .The form layout shows how the form is presented on the mobile user interface, and this influences the way a user interacts with it. The form layout is still determined by the nature of the content that is being collected. For example long survey questionnaires will have a different layout from a short mostly graphical form used by a clinician in a health facility. In addition, the designs, positions and lengths of the labels and input fields, the date format, number of columns and buttons among others all define the layout of the form .This refers to the way data is captured or entered into the form and therefore which input type is most appropriate for a given case for example check boxes, radio buttons etc. Care should always be taken not to confuse users by using many different input types in one form . In someUsers need to be guided as quickly and as error free as possible during the process of filling forms from the start by explaining restrictions in advance . This inThe form has to be submitted after filling it using a submission button . The subWe chose to use these categories because they represent some of the main activities a user is involved in when filling a data collection form. And therefore were a good basis for the selection and categorization of the questions that we included in the initial usability evaluation checklist.Heuristics for Specific Application Domains (SAD) can be generated in a number of ways, but one of the most important steps is the validation of the heuristics to ensure that they are able to do what they are supposed to do. According to Van Greunen et al.,  the valistrongly disagree, disagree, somewhat agree, agree and strongly agree with a score of 1,2,3,4 and 5 respectively. Furthermore, the developers were free to add a comment explaining their scores in addition to removing or adding to the usability evaluation questions. The developers could also suggest renaming a given question or re-locating a question to a different category.The software developers validated this initial usability evaluation set in order to refine it further and make it more usable using a validation tool. The validation tool was created as an excel file and each of the 125 usability evaluation questions was scored on a scale of 5 where the options were presented in form of a drop down list. These included The validation tool was based on 5 major assessment criteria, namely utility, clarity, question naming, categorization and measurability. The criteria was based on characteristics proposed by Van Greunen et al.  some of This tests the evaluation question’s contribution and relevance to the design of the mobile data collection form.This tests whether the evaluation question is clear and can easily be understood by the evaluator.The test was on whether the evaluation question name was appropriate.Here the test was whether the evaluation question is placed in the right category.This tested the possibility of measuring and attaching a score to the design feature using this evaluation question.Validation can be a continuous and iterative process involving novice, average and expert users. The initial assessment of the initial usability evaluation questions was performed by novice software developers in March 2017. We presented the main study objectives and the relevance of the activity the novice developers were about to undertake, after which they downloaded the checklist and the validation tool from their individual email addresses. We then trained the developers for about 10 min, after which they were given 90 min to assess the checklist and submit the completed assessment thereafter to the researchers’ email address.n = 9), District Health Information Software (DHIS2) (n = 6), Open Medical Records System (OpenMRS) (n = 5). Other applications included mUzima (n = 2), Medic Mobile Toolkit, CSpro, Survey CTO, koBo Toolbox, Survey Monkey and OpenXData. The 20 developers received an email each indicating the main study objective and the relevance of the activity they were about to engage in. On acceptance to be part of the study, the file with the assessment criteria was forwarded to them via email, and they were expected to submit it after one week.The second validation of the same evaluation questions was done by the expert developers in Uganda between October and November 2017. The expert developers had developed mobile forms for collection of health data for varying periods of time, ranging from 1 year to 8 years using software like Open Data Kit (ODK) ((Is the number of colors limited to 3–4?) was unclear and one had to read it twice to understand it. Three of the comments went on to advise on how we could improve on a given question e.g. splitting question 9  into 2 segments. One developer also felt that progress disclosure in question 38  would not be very important in a mobile view. All the 125 questions that were validated are listed in the Out of the 20 copies of validation tools that were sent out, we received 18 copies back, 3 of which were incomplete. So our results were based on the 15 complete submissions. We received a total of 9 comments from 5 developers. Five of the comments mentioned that some of the evaluation questions were not clear and therefore could possibly lead to misinterpretation or confusion. For example two developers felt that question 12 Are all abbreviated words of the same length?). Eight of the expert developers commented that it was not possible to have abbreviated words of the same length. Some questions were also found to be ambiguous e.g. (Does the tool provide informative progress disclosure when filling a form?) and (Has the skip logic been automated?). The relevance of some of the questions was also questionable e.g. . Some expert developers also pointed out that some of the design features’ performance is influenced by other factors e.g. the feedback time during data collection may be influenced by the internet speed. However this activity is rather subjective and very individualistic and therefore the developers were likely to interpret and evaluate the questions differently based on their varied experiences.All the 20 copies that were sent out to the expert developers were filled and received back after periods ranging from 2 to 4 weeks. We received more comments from the expert developers compared to the novice developers, some of which included renaming or re-categorizing the evaluation questions. For example some of the expert developers felt that questions 6, 10, 14, 15, 27, 28 and 29 needed to be put in other categories rather than the ones they were in . In addiTo generate the usability design checklist, we considered questions where 80% and above of the novice and expert developers agreed to each of the utility, clarity, question name, categorization and measurability of the questions. We then selected those questions where more than 80% of the responses indicated ‘agree’ or ‘strongly agree’ across all the 5 criteria. Because utility and measurability have higher weight than the rest of the criteria, we also considered those questions which scored above 80% in both usability and measurability. We then considered those questions where both novice and expert developers affirmed to the utility of the question. And lastly, we considered those questions where only the experts affirmed to the utility of the question. This led to a total of 64 questions. We then calculated the average of responses with ‘agree’ or ‘strongly agree’ for each question across the 5 criteria, and selected those questions with an average of 85% and above. This led to 30 evaluation questions of which 9 were categorized under the form layout, 12 under form content, 2 under the input process, 6 under error handling and 1 under form submission. These 30 usability evaluation questions are all represented in Table There were no questions where both sets of developers selected ‘agree’ to all the 5 criteria for a particular question. However, there were 11 questions in this checklist where both sets of developers selected ‘agree’ to more than one criterion for a given question. But generally expert developers affirmed to the questions based on the given criteria compared to the novice developers.We further analyzed the data based on the criteria to determine the participants’ decision for each usability evaluation question. We determined the number and the respective percentage of participants who agreed, disagreed and were neutral to a given criteria for a particular usability question for each of the experts and the novice developers.utility had 12 questions , clarity had 8 questions , and question naming had 3 questions . In addition, categorization had 6 questions  while measurability had 4 questions . We also had about 12 novice developers (80%) selecting ‘agree’ to the utility of 14 questions, to the clarity of 24 questions, to the question names of 13 questions, to the categorization of 15 questions and to the measurability of 5 questions. These results depict that majority of the questions that the novice developers agreed to were clear to them. In fact all the novice developers agreed to the clarity of question 66 . The number of novice developers who selected ‘disagree’ against questions was relatively low with the highest being 8 developers disagreeing with the question name for question 9 . There were also 40 and 27% of the developers disagreeing with the clarity and the categorization of this question respectively. The biggest percentage of disagreements (above 27%) was made up of measurability (14 questions) followed by utility (11 questions) and clarity (11 questions), and yet these are the criteria with the highest weights.For 25 usability evaluation questions 85% and above of the novice developers selected ‘agree’ i.e. measurability which was below 85%. There were incidences where all the expert developers affirmed to the criteria regarding a particular question for example utility had 5 questions , clarity had 5 questions , question name had 3 questions  while categorization had 4 questions . Question 92  however had all the expert developers agree to the utility, clarity and categorization of that question. In addition we had 7 questions  where 90% and above of the expert developers agreed on the relevance of 3 criteria and 9 questions  where 90% and above of the developers agreed on the relevance of the 4 criteria. This can be compared to questions 58 (Are inactive menu items greyed out or omitted?) and 59  where 87% of the novice developers agreed to the utility, clarity and categorization value of the questions.There were 50 evaluation questions where 85% and above of the expert developers selected ‘agree’ for all the criteria apart from,  had a high level of disagreement across all the 5 criteria, with utility having the highest diagreement of 50%.We also considered those questions where less than 50% of the novice developers selected ‘disagree’ Measurability had 29 questions, followed by utility with 13, clarity with 9, question name with 8 and lastly categorization with 8 questions. Question 24 The number of expert developers who gave high criteria scores for each usability question was higher than the number of novice developers. In addition, the scores across criteria also varied with the highest being utility followed by clarity, question name, categorization and lastly measurability, and for both sets of developers, utility scored highly while measurability scored least Fig. .Fig. 1ToThe literature search generated 125 usability evaluation questions which after validation by the novice and software developers were reduced to 30 questions. The results after the validation indicate that expert developers appeared to value the utility, clarity, question names, categorization and measurability of the questions more homogeneously as a group than the novice developers. According to both groups of developers, the questions were found to be useful, clear, with proper names and correct categorization; however both sets of developers felt that the measurability of the questions was not satisfactory.The results indicate differences in the agreement and disagreement levels with evaluation criteria of the novice and expert developers, but the general trend was the same i.e. there were some questions where both evaluator groups agreed or disagreed. For example there were 20 questions where more than 85% of the developers found 3 or 4 criteria valuable. In addition, novice developers attached great importance to the use of appropriate language, omission of inactive menus, visibility of help function, prompts and messages and lastly to the ability to switch between data collection and their work. On the other hand, expert developers attached great importance to the appropriate language use as well, visual differences between interaction objects and information objects, indication of mandatory and optional fields, visibility of the help function and the use of device information like the Global Positioning System (GPS).The variations in the levels of agreement and preferences may be attributed to differences in experience and context of operation. For example the expert developers had experience ranging from 3 to 10 years, which the novice developers did not have. Secondly the expert developers had been involved in creating MDCEFs for collection of health data in low resource settings, whereas the novice developers were more involved in mobile software development with nothing specific to Mobile data collection tools. However, in evaluation or validation it is important to have a mix of users with different skill levels e.g. the beginners or novice users, the average level users and the power or expert users to test a given product. This prevents skewing of the design requirements towards a particular group because users have varying needs based on their experience .Most of the questions the developers affirmed to were in the form content category followed by the form layout, error handling, input process and the form submission categories respectively. Measurability was hardest to assess, followed by utility and clarity for both groups of software developers. This may be attributed to the fact that it was not clear to the developers what the criterion was meant to achieve. However, we still need to define ways of measuring the achievement of a particular design feature we are testing.Expert developers had different views on more usability questions compared to the novice developers. This may be attributed to the experience they have had working on these applications such that they are able to know what is achievable or not. Secondly, some of the expert developers have had an opportunity of interacting and getting feedback from the end users especially during the training sessions, which placed them in a more advantaged position of knowing what the users may be comfortable with.Utility of a usability question was considered most important among the validation criteria and as such, the evaluation questions with high percentages of disagreement for utility were automatically eliminated from the checklist. We argue that however clear or well categorized a usability question is, it needs to be useful in the evaluation of the MEDCFs.One of the limitations was the difference in the time allocated to the 2 groups of developers; the expert developers had a lot more time to validate the initial checklist compared to the novice developers who only had 90 min. However, to the best of our knowledge, this was the first exercise of its kind where intended users get to validate the tool they will use to assess their interface designs for MEDCFs, besides the user testing of the prototype or finished product that is done with the end users. Having 2 sets of developers with varying experiences and from different contexts enriched the study because software developers only develop software with a focus on particular features which is partly the reason for the weaknesses of using Open Source Software (OSS) .The objective of this study was to compare the novice and expert developers’ views regarding usability criteria. This study generated and validated a design checklist for Mobile Electronic Data Capture Forms (MEDCFs), and was thus a way of creating awareness to what should be expected of a mobile data collection tool from the software developers’ perspective. The different results from the novice and expert developers, where we registered more affirmative results from the expert developers is an indicator of their expectations as developers. This may also be an indication of their level of engagement and knowledge of the people for whom they are creating the tools as well as the experience acquired over the years. We would thus recommend the use of more experienced developers during validation of checklists for mobile data collection tools.The checklist resulting from this study needs to be evaluated by users as software developers are not the end users of the data collection forms. We thus propose to test the effectiveness of the measure for suitability and performance based on this generated checklist, and test it on the end users (data collectors) with a purpose of picking their design requirements. Continuous testing with the end users will help refine the checklist to include only that which is most important in improving the data collectors’ experience. In addition to this first study that summarized the observations, there will be a deeper data analysis based on the collected material to determine the relationships between the criteria scores on the evaluation checklist and the developer groups.