Optical frequency combs, consisting of well‐controlled equidistant frequency lines, have been widely used in precision spectroscopy and metrology. Terahertz combs have been realized in quantum cascade lasers (QCLs) by employing either an active mode‐locking or phase seeding technique, or a dispersion compensator mirror. However, it remains a challenge to achieve the passive comb formation in terahertz semiconductor lasers due to the insufficient nonlinearities of conventional saturable absorbers. Here, a passive terahertz frequency comb is demonstrated by coupling a multilayer graphene sample into a QCL compound cavity. The terahertz modes are self‐stabilized with intermode beat note linewidths down to a record of 700 Hz and the comb operation of graphene‐coupled QCLs is validated by on‐chip dual‐comb measurements. Furthermore, the optical pulse emitted from the graphene‐coupled QCL is directly measured employing a terahertz pump–probe technique. The enhanced passive frequency comb operation is attributed to the saturable absorption behavior of the graphene‐integrated saturable absorber mirror, as well as the dispersion compensation introduced by the graphene sample. The results provide a conceptually different graphene‐based approach for passive comb formation in terahertz QCLs, opening up intriguing opportunities for fast and high‐precision terahertz spectroscopy and nonlinear photonics. Enhanced passive frequency comb operation and pulse generation in terahertz semiconductor lasers, i.e., quantum cascade lasers, are successfully demonstrated by coupling a multilayer graphene saturable absorber into a 6 mm long terahertz laser. Dual‐comb and pump–probe techniques are implemented to verify the frequency comb and pulsed operations  of graphene‐coupled terahertz lasers. Frequency combsThe general scheme employed for passive comb formation is to get a laser mode‐locked by incorporating saturable absorbers or other nonlinear reflectors in the laser cavity.FigureFigureIn this work, a relatively long laser cavity (6 mm) is intentionally used to obtain an intermode beat note frequency of ≈6 GHz . Since the terahertz photon energy is much smaller than the Fermi energy of 158 meV , the terahertz absorption in the graphene samples is an intraband transition process. Because the fabrication imperfection is accumulated during the layer‐by‐layer transferring process, the fitting curves show larger deviations from the experimental results for 30‐ and 40‐layer graphene samples. To make the graphene absorber function properly, we use a 15‐layer graphene sample in the following experiment to provide moderate linear absorption loss and sufficient nonlinearity.Both linear and nonlinear absorptions are critical for the functioning of graphene saturable absorbers. A moderate linear absorption loss can shorten the optical pulses for passive mode‐locking. However, if the linear absorption introduced by graphene absorbers is too high, the saturable absorption would hardly happen and optical pulses cannot be obtained. The linear transmission of the multilayer graphene samples in terahertz frequency range, as shown in Figure z‐scan experiment which has been used to characterize the saturable absorption of nonlinear materials.z‐scan technique is fully described in the Experimental Section and the Supporting Information. Figure z0) can be clearly observed. A theoretical fit is performed to extract the saturation parameters of the graphene sample. The average power of the QCL with a graphene‐integrated saturable absorber mirror (GiSAM) measured at z0 at 900 mA is 685 µW, which corresponds to a power density I0 = 2 W cm−2 by considering a beam waist diameter of 209 µm at z0 . The fit , an average power of 2 mW measured outside the cryostat is equivalent to 10 mW on the GiSAM in the cryostat. This can then be translated into a power density of 61 W cm−2 by considering the beam divergence and the air gap between the laser facet and the GiSAM. Finally, one can estimate a peak intensity of 610 W cm−2 or a pulse energy of 9.7 nJ cm−2. The estimated peak intensity of 610 W cm−2 is much larger than Is derived from the fit in Figure z‐scan experiment, normally the terahertz laser should be driven in pulsed mode or it is optically pulsing. However, the saturable absorption was also observed when terahertz QCLs operated in a quasi‐CW mode (with a duty cycle of 40%).z positions is shown. The nonlinear behavior of the graphene sample is related to the carrier absorption process. Once the absorption is fully saturated, the graphene will be transparent to the terahertz light. The nonlinear increase in transmission can be only observed before the graphene is fully saturated and afterward the power dependence of the transmission is linear. Since the saturation regime is easily realized in our experiment especially for z = 0, we are not able to observe the clear nonlinear increase in transmission with power. However, for z = −1 and 1 mm, as the threshold average power for saturable absorption is higher than the case of z = 0, the nonlinear behavior is observed as shown in Figure S12 in the Supporting Information.The nonlinear absorption behavior of the graphene sample is investigated using a z‐scan traces measured at 900 mA at different spots of the sample . As expected, all the z‐scan traces measured with the GiSAM‐QCL at 900 mA show almost identical behavior, which confirms that the graphene sample used in this work is uniform and the nonlinear saturable absorption is obtained when the sample is illuminated by the GiSAM‐QCL.Moreover, further experiments are implemented to investigate the saturable absorption of the graphene sample. In Figure S2 in the Supporting Information, the light–current curves for the QCL with Si mirror and GiSAM measured in the constant current mode are shown. We can see that the slope efficiency changes with increasing the current for the QCL with GiSAM, while it remains constant in the entire current dynamic range for the QCL with a Si mirror. This infers the nonlinearity introduced by the graphene sample. To verify that the saturable absorption observed in Figure The mode stability of GiSAM‐QCLs is first studied employing an electrical intermode beat note measurement  which has been widely used to characterize the mode coherence of terahertz lasers by recording the down‐converted beating signal of optical modes.FigureWe further monitor the long‐term frequency stability of the QCL modes by switching on the “max‐hold” function of the spectrum analyzer to record and store the spectral maxima during the time duration (3 min) of the measurement. Figure fb1 and fb2, are separated by 14 MHz. Here, we intentionally separate the two laser combs by 1.6 mm  of the Fourier transform infrared spectrometer. The down‐converted dual‐comb spectrum shown in Figure Figure igure −1  GHz of tigure −1  GHz of tigure −1  GHz of tWe next compare the dual‐comb spectrum of FP‐QCLs with that of GiSAM‐QCLs. Similarly with Figure FigureFinally, to further prove that the GiSAM‐QCL is pulsing, we perform a terahertz pump–probe experiment. The experimental data shown in Figures The other origin of the enhanced passive frequency comb operation in the GiSAM‐QCL is the dispersion compensation introduced by coupling the graphene sample that slightly changes the reflectivity of the GTI mirror. Theoretical studies show that the graphene is not able to significantly change the terahertz reflectivity at the air/Si interfaceCompared with existing methods such as active mode‐locking or using passive mirrors to compensate dispersions, the passive way of using a graphene absorber to achieve frequency comb operation shows two advantages/merits. First of all, the passive technique in this work can get rid of complex electronics and optics for intracavity loss modulation and active mode stabilization, and/or mirror designs and fabrications for compensating the group velocity dispersions in QCLs. Furthermore, the microwave impedance mismatching is not necessary to be taken into account in the passive scheme employed in this work. Thus, the passive comb system is more compact than the active terahertz QCL combs. Second, the passive scheme (in principle) allows the generation of much shorter pulses than the active one because a saturable absorber can modulate the losses much faster than electronic modulators. However, this doesn't mean that the absorber should be fast to achieve passive mode‐locking. We only require the absorber to respond fast to the leading wing of the pulse. Sometimes, a slow absorber is even better for achieving self‐starting mode‐locking in lasers.z‐scan measurements for a 15‐layer graphene sample yield a saturation peak intensity of 7.3 ± 0.12 W cm−2 and a saturable absorption coefficient of 0.21 ± 0.01 at 4.2 THz. The on‐chip dual‐comb has been successfully performed to prove the passive comb operation in the terahertz laser. Furthermore, a pulse width of 16 ps has been obtained from a terahertz pump–probe experiment, which directly proves the pulse generation in GiSAM‐QCLs. The possible origins of the enhanced passive frequency comb operation and pulse generation, i.e., saturable absorption and/or dispersion compensation introduced by the graphene sample, are discussed in detail. The highly stabilized passive frequency comb offers a powerful tool that can be used in terahertz metrology applications. For instance, the dual‐comb technique enables fast spectroscopy with Doppler‐limited resolution. The ultrashort terahertz pulses generated from GiSAM‐QCL combs could also find promising uses in studying the carrier dynamics of various materials, such as 2D materials and III‐V semiconductors.In summary, we have demonstrated a terahertz frequency comb that exhibits significant self‐mode stabilization owing to the incorporation of a multilayer graphene‐integrated saturable absorption mirror into a terahertz QCL. The intermode beat note linewidth is as narrow as 700 Hz. Nonlinear terahertz Terahertz QCL: The terahertz QCL used in this work was based on a hybrid active region structure that combines the bound‐to‐continuum optical transitions and the resonant phonon scattering for depopulation. The layer sequence of one period for the QCL active region with 76 cascade periods, from the injection barrier, is 4.1/3.8/1/23.6/1/13.8/2.1/11.8/3.1/9.6/3.1/8.7/3.1/7.7/3.1/17.2/3.4/14.8 nm, where Al 0.25Ga0.75 As layers are listed in bold, GaAs layers in normal font, and the 17.2 nm thick GaAs layer (in underlined font) is doped to 2 × 1011 cm−2. The entire QCL active region was grown using a molecular beam epitaxy system on a semi‐insulating GaAs substrate. The grown wafer was then processed into single plasmon waveguide laser ridges using traditional GaAs processing technology, such as optical lithography, electron beam evaporation, chemical and dry etching, lift‐off, etc. The ridges were cleaved into laser bars with cavity lengths ranging from 2 to 6 mm and then indium‐soldered onto copper bases (heat sink). Finally, the QCL device was mounted onto a cold finger of a cryostat for electrical and optical measurements.−1 (3 GHz).To measure the CW power of the terahertz QCL, two off‐axis parabolic mirrors were used for collecting and focusing the terahertz light onto a terahertz sensor Ophir, 3A‐P THz), which was connected to a power meter for reading the CW power value. The power value shown in Figure S1b in the Supporting Information is the measured power without any calibration of light collection efficiency, window transmission, water absorption, or mirror reflection. The emission spectra shown in Figure A‐P THz, Multilayer Graphene Fabrication and GiSAM Integration: The multilayer graphene samples were fabricated through a layer‐by‐layer transfer technique of monolayer graphene film grown via chemical vapor deposition. Graphene layer growth was carried out in a quartz tube at reduced pressure with a copper foil  as the catalyst substrate. During the process, the copper foil was heated up to 1000 °C and kept for 1 h under a 10 sccm flow of hydrogen (≈400 mTorr) to reduce the oxide presence, increase the grain size, and ensure a smooth surface for graphene growth. Next, methane gas with a beam flux of 10 sccm was introduced into the chamber for the graphene synthesis, and the gas pressure was maintained at ≈750 mTorr for 30 min. Following that, the grown sheet was cooled to room temperature in an atmosphere of hydrogen. The transfer of the graphene films onto the double‐side‐polished Si mirror (with a diameter of 10 mm) was realized by utilizing the conventional float‐transfer method. A polymethyl methacrylate resist was used as the protective layer, and the iron chloride etchant was used to etch away the copper substrate. The number of graphene layers on the desired substrate was controlled by the number of times the transfer was repeated. It is worth noting that additional loss was introduced because of the interfacial dangling bonds, and graphene film quality degrades as the number of monolayer graphene transferred increases.c/2ln, where c is the speed of light in vacuum, l = 410 µm is the length of the air gap , and n is the refractive index of air. Since the modulation period was much larger than the emission spectral range shown in Figure The multilayer graphene and Si mirror constructed a nonlinear reflector, GiSAM, which was then coupled to the 6 mm long terahertz QCL using a sample fixture as shown in Figure S1e in the Supporting Information. It has to be clarified that by coupling the GiSAM into the QCL compound cavity, an etalon consisting of the air gap and the air/Si interface was introduced, which might result in loss modulation and mode selection in the QCL. The modulation period was calculated to be ≈360 GHz by following the formula Δν = Open‐Aperture z‐Scan: As shown in Figure S9a in the Supporting Information, the terahertz light emitted from the GiSAM‐QCL was collimated and then focused onto the graphene sample by a pair of off‐axis parabolic mirrors with a reflected focal length of 101.6 mm. The sample was placed on a motorized micrometric stage  moving along the optical axis (z). After being transmitted through the graphene sample, the light was collected and focused onto a terahertz power detector using another pair of parabolic mirrors, and the power value was read using a power meter . The beam focal spot size (w0) was measured to be 209 µm, with a Rayleigh length (zR) of 600 µm. The open‐aperture z‐scan was performed at different QCL drive currents  by recording transmittance versus sample position along the z‐axis. The step size of the stage was set to 0.4 mm. For each QCL drive current, the measurement of the transmitted terahertz intensity was repeated 10 times and the mean value for fitting was taken. The transmittance of the 0.3 mm thick Si‐substrate was also recorded and fitted with a third‐order polynomial function, which was used as a baseline for data normalization. Details of the fitting procedure can be found in the Supporting Information.Intermode Beat Note Measurement: The intermode beat note spectra were measured electrically using a spectrum analyzer with the assistance of a broadband Bias‐T . To extract the high‐frequency signal from the laser cavity, a microwave transmission line as shown in Figure Marki, BT‐0026. ToOn‐Chip Dual‐Comb: The on‐chip dual‐comb device that was investigated here consisted of two terahertz QCL combs, one of which acted as a local oscillator (LO) comb (Comb1) and the other one was a sample comb (Comb 2)  of both combs should be slightly different. Otherwise, no dual‐comb spectrum can be observed. Once two single narrow intermode beat notes as shown in Figure Terahertz Pump–Probe: The terahertz pump–probe experiment was carried out by exploiting the nonlinearity (saturable absorption) of the graphene sample. As the graphene film can be saturated by the terahertz light (see Figure The authors declare no conflict of interest.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.


Classroom questioning can be considered a key factor in the promotion of student engagement. This case study explored classroom questioning practices and perceptions of a group of 47 first-year law students and their teacher. Eight lessons of 90 min were observed and audio-recorded and afterward the students and the teacher answered a questionnaire. The teacher was also interviewed. Researchers examined the number and type of questions asked by the teacher and by the students in the classroom and analyzed the students’ and the teacher’s perceptions about the importance of classroom questioning. Results indicated that the teacher and most students consider questioning important or very important for student learning. The number of questions posed by students as opposed to by their teacher was not balanced, as the teacher was responsible for 93% of the questions. The analysis of the type of questions posed by the teacher and by the students showed a predominance of low-order questions. Therefore, classroom questioning in this case study did not seem to promote students’ autonomous thinking. The current study suggests the importance of examining the teacher and students’ patterns of questioning together, analyzing its similarities and discrepancies. Colleges and universities are challenged to adopt teaching and learning methods that promote students’ agency and autonomy, as well as high levels of thinking skills and student engagement . ClassroResearch shows the relevance of questioning as a teaching technique that contributes to engaged learning and the promotion of deeper-level thinking by students . QuestioIn different levels of schooling, several studies emphasize the lack of high-order questions and the predominance of factual or recall-type questions, as well as procedural questions . The stiLiterature has highlighted the importance of teacher and student questioning for both teaching and learning , yet theM = 19.50, SD = 2.15) and their respective teacher  in a Portuguese private urban university that offers different study areas and degree levels. Among these students, 63.8% were attending college for the first time.Participants were 47 first-year law students  and student questioning (one item) in the classroom. The scale for the two items contained five possible answers ranging from 1, meaning After the observational and questionnaire data collection, a semi-structured interview was conducted with the teacher to give him feedback about the findings and explore possible explanations to the results. Interview questions referred to the importance of teacher’s and students’ questioning in the classroom and its main functions. The teacher presented his explanations for the observed data and reflected on the kind of questions he used to pose to his students.All the elements of this case study contributed to the purpose of the study. Besides the analysis of the typology of the questions posed by the teachers and by the students, the questionnaire allowed researchers to analyze participants’ perceptions about the value they attributed to teacher’s and students’ questioning, and the interview enabled them to collect relevant data about teacher perceptions of classroom questioning, reflecting from data from his own classes.We carried out this study following the recommendations from the ethics committee of the University of Minho. Participants gave written informed consent to participate in the research in accordance with the Declaration of Helsinki. Participants were informed about the objectives of the investigation and confidentiality and anonymity were assured. In addition, participation in our study was voluntary and researchers informed students about data usage.The number and the type of questions asked both by the teacher and by the students were analyzed. All issues recorded, after analysis, were categorized. These categories were created according to a semi-inductive process, keeping in mind the typologies consulted in the literature review e.g., . The selThe perceptions of the teacher and the students about the importance given to classroom questioning were analyzed (descriptive statistics) alongside the qualitative data collected through the teacher interview .pseudo questions when there was no expectation of a response and no waiting time was given after the question. Procedural questions referred to course functioning. Academic questions referred to the learning material and intended to support students’ learning and to build or assess students’ knowledge.The teacher’s and the students’ questions were classified in three categories see . Questiopseudo questions (50.0%), 356 academic questions (39.0%), and 101 procedural questions (11.1%). The average number of teacher questions per class was 57.1 (excluding pseudo questions).Considering the eight lessons observed see , the teapseudo questions category was the most prevalent (63.4%), followed by the emphasizing questions (16.1%), questions designed to check clarity of the exposition (15.2%), questions identifying students’ doubts (6.3%), and questions relating contents (0.2%). Content related questions, either open or closed, with the objective of constructing knowledge and helping students to think autonomously, were not observed. The teacher’s lectures could be characterized as utilizing an expository method, in which questions were mainly used to emphasize the discourse, as the teacher frequently answered his own questions . The next most common categories were monitoring students’ attendance questions (20.8%), questions assessing individual students’ knowledge (19.2%), emphasizing questions (8.8%), questions checking exposition clarity (4.5%), questions identifying students’ doubts (2.6%), and finally content related questions: closed questions (1.5%) and open questions (1.3%).Throughout the practical classes, In practical lessons, the teacher opted for assessing students’ knowledge orally, asking questions about the learning material. Researchers observed an intense interaction between teacher and students, as students were individually questioned, in the context of a continuous assessment modality . In spitIn comparison to lectures, in practical lessons, content related questions, while they only represented a small percentage of the amount of total questions observed, were a novel addition. Moreover, all the content related questions corresponded to low-order questions, namely remembering and understanding, the first two categories of Bloom’s revised taxonomy . Thus, iIn the eight lessons observed, students asked 69 questions, namely academic questions (94.2%), and procedural questions (5.8%). The average number of students’ questions per class was 8.6.In both lectures and practical lessons, the content related open questions were predominant , followed by content related closed questions (39 and 32%), and by questions clarifying students’ assessment conditions (7 and 4%). The content related questions were mostly requests for clarification, concept definitions or repetition of certain concepts. Also in this case, questions posed by the students were low-order questions, namely Bloom’s taxonomy remembering and understanding categories .The findings also revealed that the number of questions asked by students in lectures and in practical lessons was higher before the exam and decreased after the exam, showing that students’ participation grows when they study more intensively. These data reinforce the importance of continuous assessment practices that promote regular academic engagement.Data from the questionnaire showed that the teacher and the students agreed that classroom questioning is important or very important for student learning and that both attribute more importance to their own questions.pseudo questions). These findings are congruent with prior studies conducted in this area (e.g., Through examination of the total number of questions asked in the eight classes, it was concluded that the number of questions asked by the teacher and the students was unbalanced: the teacher was responsible for 86.9% of the questions (excluding ea e.g.,  pointingThe analysis of the type of questions posed by the teacher and by the students revealed important similarities: the predominance of low-order questions, eliciting remembering and understanding processes; and the focus on basic comprehension of the concepts. In both cases, classroom questioning did not seem to be used in order to promote students’ autonomous thinking. These findings are consistent with literature e.g., .In the interview, teacher showed that his central concern was to transmit the contents in a very well organized and clear manner to the students, mainly because his students were first-years and were learning new concepts for the first time. This concern is consistent with the type of questions posed by the students. As referred by Overall the current study suggests the importance of examining the teacher’s and the students’ patterns of questioning and discussing their similarities and discrepancies. The findings of this case study suggest a mirror effect between the patterns of these two actors in the classroom. It could be important to explore the role of the modeling exerted by the teachers on their students.The findings of the current study, while limited, suggest interesting educational implications. There is a need for learning experiences aiming at promoting metacognition processes in college classes, allowing teachers and students to explore their perceptions about teaching and learning conceptions and approaches, student classroom participation and questioning beliefs, functions, and practices . Higher The datasets for this study will not be made publicly available because the researchers reported in the informed consent that the data would only be used by the research team who conducted the study.This study was reviewed and approved by the Ethics Committee of the University of Minho. All research participants provided written informed consent in accordance with the Declaration of Helsinki.LR and PR contributed to the conception and the design of the work. LR, PR, and IM were responsible for data collection, analysis, and interpretation of data. LR wrote the manuscript with valuable inputs from the remaining authors. RSC and IM made important intellectual contributions for the manuscript. All authors agreed for all aspects of the work and approved the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


N,N-dimethyl acrylamide) and poly(ethylene oxide) blocks and are used as humidity sensitive media. Instead of using glass or opaque wafer for substrates, polymer thin films are deposited on Bragg stacks and thin (30 nm) sputtered Au–Pd films thus bringing color for the colorless polymer/glass system and enabling transmittance measurements for humidity sensing. All samples are characterized by transmittance measurements at different humidity levels in the range from 5% to 90% relative humidity. Additionally, the humidity induced color change is studied by calculating the color coordinates at different relative humidity using measured spectra of transmittance or reflectance. A special attention is paid to the selection of wavelength(s) of measurements and discriminating between different humidity levels when sensing is performed by measuring transmittance at fixed wavelengths. The influence of initial film thickness, sensor architecture, and measuring configuration on sensitivity is studied. The potential and advantages of using top covered Bragg stacks and polymer/metal thin film structures as humidity sensors with simple optical read-outs are demonstrated and discussed.Thin films with nanometer thicknesses in the range 100–400 nm are prepared from double hydrophilic copolymers of complex branched structures containing poly( Humidity is present everywhere and is one of the mostly important physical parameters in our lives. The accurate monitoring of humidity is significant in semiconductors, electronics, food processing, and pharmaceutical industries where the quality of products is influenced sufficiently by the humidity. Humidity control is also essential in museums or archives in order to ensure the correct storage of artworks. Further, monitoring of relative humidity in office environments is in favor of human comfort and health and helps achieve hygienic conditions.The perfect humidity sensor should have high sensitivity, fast response, long-term durability, low cost, easy detection, and wide dynamic range, i.e., it should operate over a wide range of humidity. The established technology nowadays is an electrical measurement of humidity  where thIn recent years, optical sensing of humidity has gained increasing interest and great research efforts have been put in this field ,5. OpticCost effective and relatively simpler approaches are color sensing of humidity or monitoring of appropriately chosen optical parameters that change with humidity. In the first approach the detection is performed by visual inspection of color, or color monitoring by an inexpensive camera ,10,11. DPolymers attract considerable interest as optical sensing materials due to the advantageous features such as flexibility, light weight, ease of processability, useful mechanical properties, and compatibility with oxides and ceramics ,3. In geIt is already demonstrated that thin films with submicron thicknesses exhibit faster swelling than bulk hydrogels . We haveIn this paper, for detection of humidity we use transmittance measurements because they are simpler, more accurate, and less expensive compared to reflectance measurements. Moreover, observing sample color in transmittance mode, i.e., using the light transmitted through the sample, could overcome to a great extent the ambiguity related to the angle dependence of the color when reflectance mode is used. We study two sensor configurations. In the first one we deposit the sensitive film on top of the Bragg stack, instead of incorporating it in the stack’s backbone, thus overcoming possible issues of incompatibility that may arise when organic and inorganic materials are used for the stack. The second sensor configuration is comprised of a thin metal sublayer deposited on glass substrate and top-covered with the sensitive polymer film. The metal sublayer has dual purposes: to provide color for colorless polymer film and to increase the optical contrast between the polymer and glass substrate that have almost the same refractive indices (around 1.5).N,N-dimethyl acrylamide) (PDMA) and poly(ethylene oxide) (PEO) segments [In this study we prove the concept of utilization of top covered Bragg stacks and polymer/metal thin films as optical sensors for humidity. As humidity sensitive media we use thin films of previously developed multiblock copolymer with branched macromolecular architecture comprised of poly was purchased from Sigma–Aldrich  and purified from inhibitor by passing through a column of basic alumina before use. Poly(ethylene oxide) of molar mass 2000 g·mol–1 (PEO2000) was supplied from Fluka . Initiator diammonium cerium (IV) nitrate (NH4)2Ce(NO3)6 and crosslinker poly(ethylene glycol) diacrylate of reported average Mn 575 (PEGDA) were purchased from Sigma–Aldrich as well. Other solvents and reagents were of standard laboratory reagent grade and used as received.The monomer N,N-dimethylacrylamide (DMA) in deionized water was carried out using ammonium cerium (IV) nitrate as the initiator. Poly(ethylene oxide) and poly(ethylene glycol) diacrylate were implemented as a hydroxyl functionalized initiating moiety and as a cross-linker, respectively. The polymerization was carried out for 3 hours at 35 °C in nitrogen atmosphere under vigorous stirring and terminated by diluting the reaction mixture with methanol (1:1 volume ratio). The copolymer solution thus obtained was used for thin polymer film deposition without further isolation or purification. In order to control the thickness of the films, the copolymer solution was diluted to the appropriate concentration by using a water/methanol mixture (1:1 volume ratio). Chemical structure of the copolymer is schematically presented in The synthesis of the copolymer is described in detail in our previous papers ,27. Brie−1 and duration of 60 s using polymer solutions with concentrations of 4, 6.25, 7.3, and 8.3 g L−1, respectively. A post deposition annealing at 180 °C for 30 min in air was applied to all films. Silicon wafers, Bragg reflectors, and Au–Pd coated glasses were used as substrates. Five- and seven-layer Bragg reflectors were prepared on glass substrates by alternating deposition of sol-gel Nb2O5 [2 [2O5 films [−2 mbar using a Mini Sputter Coater SC7620 system. The thickness and diameter of the target were 0.2 and 57 mm, respectively. The purities of gold and palladium were 99.99% and 100%, respectively, and Au:Pd ratio was 80:20. The Au–Pd layers’ thickness was determined using mechanical profilometer Talystep  and confirmed by 3D optical profiler .Thin polymer films with thickness of 100, 200, 300, and 400 nm were deposited by the spin-coating method at a rotation speed of 4000 sel Nb2O5  and SiO2Nb2O5 [2  films orRefractive index, extinction coefficient, and thickness of polymer films were calculated using previously developed two-stage nonlinear curve fitting of reflectance spectra measured with UV-VIS-NIR spectrophotometer  at normal light incidence . The accIndeed, the reflectance and transmittance spectra of the obtained thin polymer films were registered in at least three consecutive experiments for each film sample. As the reflectance and transmittance data points derived from the subsequent experiments were practically equal, standard deviations were not added to the figures in the manuscript. The reported accuracy of the spectrophotometer measurement is 0.1% for transmittance and 0.26% for reflectance.S, measured in % per % RH, was calculated according to the following equation:R  is the change of sensor’s reflectance (or transmittance) in % which is provoked by the alteration of relative humidity from RH1 to RH2.The sensitivity of the sensors RH), measured in % RH, was calculated by Equation (2):errR  is the experimental error (measurement accuracy) of R or T and S is the sensitivity, calculated by Equation (1).The accuracy/resolution of detection , i.e., the swelling, is calculated according to Equation (3):d70% and d5% are the calculated film thicknesses at relative humidity of 70% RH and 5% RH, respectively.Thin polymer films with different thicknesses (from 100 to 400 nm) were deposited on Si-substrate and exposed sequentially to relative humidity of 5% RH and 70% RH. Reflectance spectra of the films were measured at both humidity levels and optical constants and film thicknesses were determined using non-linear curve fitting . Reflectd increases with thickness: 26%, 73%, and 97% for polymer films with initial thicknesses of 100, 200, and 300 nm, respectively. For the film with initial thickness of 400 nm a small decrease in Δd is observed (84%) compared to those of an initial thickness of 300 nm. The possible reason for the thickness dependence of the swelling is the adhesion to the substrate. It could be expected that the influence of substrate adhesion is the strongest for the thinnest film and decreases gradually with thickness. This explains the fourfold increase in swelling of polymer film with a thickness of 300 nm, as compared to this with a thickness of 100 nm.max) when the humidity in the measuring cell goes continuously from 5% RH to 80% RH. max is selected as the wavelength at which the humidity response is the strongest. Considering that the transmittance and reflectance of thin films are non-linear functions of film thickness  and thick (300 nm) polymer films we deposited them on glass substrate and monitored the temporal change of transmittance at fixed wavelength . This is beneficial for using the film as a sensing medium for a wide range of humidity. Similar linear dependence is obtained for 200 nm thick film in 40–90% RH. The expansion of the thickest film is exponential with the increase of the relative humidity and changes from 314 nm at 5% RH to 965 nm at 90% RH. The overall change of film thicknesses is 31%, 127%, and 207% for 100, 200, and 300 nm thick films, respectively.The swelling dynamic, i.e., the growth of film thickness with relative humidity, is shown in Considering the substantial change in thickness with humidity, we could expect also considerable change of color. If it turns out this is the case, the films could be used as color indicators for humidity. However, first the evolution of color with humidity should be investigated. Using the measured reflectance spectra of films at different humidity we calculated the CIE color coordinates  and plotThe color of the thinnest film is almost the same for RH values in the range from 5% to 40% RH and starts to change gradually from dark blue to light blue for RH higher than 40%. The color coordinates are well separated in the color space thus enabling color sensing of humidity in the range 40–90% RH. Although the thickness values at 5% RH and 90% RH are quite different for thicker films, 202 and 456 nm, respectively, the colors of the films at these humidity levels are very similar . RemainiThe main drawback when color sensing is used is the dependence of the color on the viewing angle. The observer cannot be sure whether the color change is due to the humidity change or it is because of his position during the color inspection. It is well known that reflectance and transmittance are functions of the incident angle of the light. Because the color perception of the observer is associated with reflectance or transmittance spectra, their change with incident angle of light results in different colors observed at different viewing angles. For example T or reflectance, R) at a fixed wavelength, λmax. As mentioned above, because T and R are nonlinear functions of n, k, d, and λ, the selection of λmax is not a trivial task and has an important role in detection. For example, if we pick a wavelength of 550 nm for λmax for polymer with a thickness of 100 nm  and the ambiguities associated with angle dependence of color are overcome to the great extent. However, to make transmittance measurements possible, a transparent substrate is required. The cheapest option is to use glass or plastic substrates but in our case they are not suitable because their refractive indexes match the refractive index of polymer thin film and the accuracy drops substantially. Besides, no color will be observed.Recently we have shown ,32 that Another advantage of using a Bragg stack as a transparent substrate is illustrated in T) of 5-layer Bragg stacks covered with polymer films with different thicknesses in the range 100–400 nm is shown in T increases with the thickness of the polymer film reaching a steady state for thicknesses higher than 250 nm. Therefore, in the next step of our investigation we use polymer films with a thickness of 300 nm and deposit them on four different Bragg stacks comprised of five and seven alternating layers of Nb2O5/SiO2 and dense/mesoporous Nb2O5. Our previous results have indicated that the characteristics of stacks, such as operating wavelength, number of layers, optical contrast, etc., do not substantially influence the sensitivity [2O5 and SiO2 layers. In order to achieve the highest sensitivity, we optimized the polymer film thickness through theoretical modeling. The calculated humidity induced change in transmittance  for measuring T are 650 and 730 nm , considering 0.1% experimental error in T.The detailed study of the spectra reveals that the most appropriate wavelengths  and the observer will need additional measurements to discriminate between them. We have shown that this could be overcome if color sensing is used simultaneously. Although the sensitivity and resolution are high, an ambiguity exists due to the same T decreases with the thickness of the Au–Pd film from 74% to 33% . For further experiments we selected Au–Pd film with a thickness of 30 nm because it has an average transmittance of 50%. The change of T spectra with humidity is shown in R of single film on opaque substrate . The respective resolutions are 0.7% RH and 0.3% RH. As in the case above, when using samples comprised of polymer and metal film, ambiguity in the sensing also exists due to the same T-values at different RH. In this case, the discrimination between different RH values is also possible if the color of the sample is monitored simultaneously. The dependence of T. As an example, the values of T measured at wavelength of 430 nm are presented as blue numbers in T values at 700 nm.Instead of observation of color, the discrimination between RH with the same transmittance values is possible if a second wavelength is used for monitoring of The obtained results are summarized in As mentioned in the Introduction section, the most widely spread and sensitive technology for humidity sensing is fiber optic sensing. Unfortunately they have disadvantages of relatively high cost of optical equipment, particularly spectrometers and optical spectrum analyzers, and complicated preparation of sensitive elements, especially in the case of side-polished optical fibers. The sensitivity of fiber optic sensors is calculated as nm per % RH because usually for detection the wavelength shift of the signal dip is monitored. The sensitivity values reported in the literature vary in a broad range from 0.023 nm/% RH to 1.01 nm/% RH . In ordeN,N-dimethylacrylamide)-based copolymer with optimized thickness. The sensitivity and resolution of detection as well the dynamic range were compared to the case of single film on silicon substrate. The widest dynamic range of sensing (5–90% RH) was obtained for polymer with a thickness of 100 nm deposited on silicon substrate. The sensitivity was 0.13%, while the resolution was 2% RH. The disadvantage to this method is that, in this case, monitoring of reflectance should be applied. The optimal sensing configuration was a 300 nm thick polymer film deposited on a Au–Pd thin film with a thickness of 30 nm and measuring transmittance signal at two appropriately chosen wavelengths. A sensitivity of 0.14 % was achieved for the 25–70% RH range, which increased to 0.39% for higher humidity (70–90% RH). Relative humidity of 0.7% and 0.3% could be resolved, respectively. There was a narrowing of the dynamic range (25–90% RH), which is acceptable. When a top-covered Bragg stack was used as a humidity sensor a sensitivity of 0.37% and resolution of 0.3% RH could be achieved for a relative humidity range of 40–80% RH.The concept of using polymer top-covered Bragg stacks and polymer/metal thin film structures for optical sensing of humidity was verified and confirmed. The sensing medium was a thin film of branched poly(


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.