Despite nearly a half century of studies, it has not been fully understood how pulmonary alveoli, the elementary gas exchange units in mammalian lungs, inflate and deflate during respiration. Understanding alveolar dynamics is crucial for treating patients with pulmonary diseases. In-vivo, real-time visualization of the alveoli during respiration has been hampered by active lung movement. Previous studies have been therefore limited to alveoli at lung apices or subpleural alveoli under open thorax conditions. Here we report direct and real-time visualization of alveoli of live intact mice during respiration using tracking X-ray microscopy. Our studies, for the first time, determine the alveolar size of normal mice in respiration without positive end expiratory pressure as 58 ± 14 (mean ± s.d.) μm on average, accurately measured in the lung bases as well as the apices. Individual alveoli of normal lungs clearly show heterogeneous inflation from zero to ~25% (6.7 ± 4.7% (mean ± s.d.)) in size. The degree of inflation is higher in the lung bases (8.7 ± 4.3% (mean ± s.d.)) than in the apices (5.7 ± 3.2% (mean ± s.d.)). The fraction of the total tidal volume allocated for alveolar inflation is 34 ± 3.8% (mean ± s.e.m). This study contributes to the better understanding of alveolar dynamics and helps to develop potential treatment options for pulmonary diseases. Pulmonary alveoli, the elementary gas exchange units of the mammalian lungs, continuously inflate and deflate during respiration. This dynamic behavior of the alveoli significantly influences pulmonary function and stability1246homogeneous or heterogeneous in live, breathing mammalian lungs. In addition, the fraction of the total tidal volume allocated for the inflation of the alveoli versus the non-alveolar parts of the lungs  remains undetermined101112The average size of the alveoli in live mammalian lungs (which is affected by the changing lung volume associated with breathing) remains undetermined, although this information is fundamental for understanding alveolar dynamics. When mice are sacrificed and the lungs are excised, the size of alveoli undergoes significant changes due to the drastically altered conditions 8Real-time imaging of the alveoli is essential for determining the alveolar dynamics during respiration but it has been hindered by active lung movement151616171883) regions2425In this study, we investigate alveolar dynamics not only in the lung apices but also in the bases in live intact mice during respiration, using tracking X-ray microscopy (TrXM II). X-ray imaging based on phase contrast and strongly collimated synchrotron X-rays2022Visualization of alveoli described in a previous reportIn the present study, we developed TrXM II  that allreal-time the size and the degree of inflation of individual alveoli in the lung bases during respiration.Indeed, TrXM II enabled us to track individual alveoli in the lung base .heterogeneous inflation from zero to ~25% in size (P = 0.261) for the RL bases, and to 8.7 ± 6.6%  for the LL bases.Interestingly, the individual alveoli in normal mice show  in size . The ave in size , slightlP = 0.034) in the RL bases and further to 5.5 ± 0.9 μm  in the LL bases.Depending on the lung region, the lung movement is significantly different. The smallest is for RU (86 ± 18 μm (mean ± s.e.m.)), increased for LU (214 ± 43 μm (mean ± s.e.m.)) and RL (423 ± 67 μm (mean ± s.e.m.)), and the largest is for LL (513 ± 76 μm (mean ± s.e.m.)), as demonstrated by the regional displacements of the pleural surfaces during respiration in Despite recent improvements in the experimental techniques used for studying alveolar dynamics, previous works have been limited to studying alveoli at the right upper lung apices that have minimal lung movement, subpleural alveoli under open thorax conditions, or alveoli that have been removed from the body and processed for histology. The strength of TrXM II imaging we use in this study is that it is applicable to dynamic studies of thick living organs in active movements. At the same time it has high spatial and temporal resolution. Here we demonstrate that the high penetrating power of synchrotron hard X-rays enabled us to directly visualize and accurately measure individual alveoli in live intact mice not only in the lung apices but also at the basal lung area during ventilation.ave = 1.8 ± 0.2 × 10−5 μL.  at functional residual capacity (FRC) and heterogeneous alveolar inflation by 6.7 ± 4.7% (mean ± s.d.) during respiration. The fraction of the total tidal volume allocated for alveolar inflation was estimated as 34 ± 3.8% (mean ± s.e.m). TrXM analysis of the alveoli can open the way to various studies of alveolar dynamics in live intact animals, with future promise to contribute to a better understanding of emphysema and pulmonary edema, and other diseases related to alveolar dysfunction, for instance, VILI in ARDS.The methods were carried out in accordance with the approved guidelines.−1) was injected into peritoneum. Tracheotomy was then performed, followed by the insertion of a catheter . Ventilation of mice was carried out with room air via the catheter using a ventilator (Inspira-Advanced Safety Ventilator-Pressure Controlled (ASVP), Harvard Apparatus, USA). Physiological respiratory conditions were chosen with an inspiration/expiration ratio of 1:2, a tidal volume of 160 μL/respiration, and a respiratory rate of 100 breaths/min without positive end expiratory pressure. Each mouse was placed in an acrylic tube and fastened in a vertical position for X-ray imaging. After the imaging experiments, all the mice were alive.All experimental protocols were approved by the SPring-8 Experimental Animals Care and Use Committee. Eight-week-old SPF pathogen-free nude mice  were examined. For anesthesia, sodium pentobarbital (50 mg kghttp://www.spring8.or.jp). The X-ray beam produced by an in-vacuum undulator was monochromatized to 15 keV by a double crystal monochromator, and then transported into the experimental hutch, which was located 98 m from the radiation source. By the high coherency of the monochromatic X-ray, alveolar boundaries, the boundaries between tissues and air, of live intact mice can be clearly visualized by the edge-refraction enhancement44Real-time X-ray imaging experiments were performed at the RIKEN Coherent X-ray Optics beamline (BL29XU) at SPring-8  to 1 mm × 1 mm so that it would amply cover the maximum movements of the lung bases of mice while providing the high spatial and temporal resolutions of 500 nm and 8 ms, respectively, to resolve and track individual alveoli during respiration. For this purpose, we applied a CMOS camera ) in 12-bit (dynamic range) with a maximum frame rate of 86400 f/s. To improve the spatial and temporal resolution, we then adapted the miniaturized conversion (x-rays to visible lights) systemAlveolar size was measured both at the end of expiration and at the end of inspiration from 2-D microradiographs. Individual alveoli were first identified with their individual movement paths in 2-D microradiographs during respiration similar to our previous studyTo confirm the validity of 2-D measurement, the alveolar sizes measured in 2-D and 3-D images were compared. For this, microtomography was performed for a right lower base, using TrXM II with a high-sensitivity camera , with an exposure time of 20 ms and a resolution of 590 nm/pixel. Each image was taken at the end of expiration during 180° rotation . 20 alveData were presented as mean ± s.d. or mean ± s.e.m. P-values were determined by performing a two-tailed t-test with data obtained from the right upper apices.S.C., N.K. and J.K. performed experimental work and data analyses. Y.K. and T.I. supported to run synchrotron X-ray imaging experiments in SPring-8. C.K.R. and A.T. performed data analysis and interpretation. J.H.J. supervised the work. J.H.J. and C.K.R. helped to design the experiments. S.C., N.K., J.H.J. and A.T. wrote the manuscript and prepared figures 1–4. All authors reviewed and edited the manuscript.Supplementary InformationSupplementary Video 1Supplementary Video 2


Arthrospira platensis biomass was used in order to obtain functional lipophilic compounds through green extraction technologies such as supercritical carbon dioxide fluid extraction (SFE) and microwave-assisted extraction (MAE). The temperature (T) factor was evaluated for MAE, while for SFE, pressure (P), temperature (T), and co-solvent (ethanol) (CS) were evaluated. The maximum extraction yield of the obtained oleoresin was (4.07% ± 0.14%) and (4.27% ± 0.10%) for SFE and MAE, respectively. Extracts were characterized by gas chromatography mass spectrometry (GC-MS) and gas chromatography flame ionization detector (GC-FID). The maximum contents of functional lipophilic compounds in the SFE and MAE extracts were: for carotenoids 283 ± 0.10 μg/g and 629 ± 0.13 μg/g, respectively; for tocopherols 5.01 ± 0.05 μg/g and 2.46 ± 0.09 μg/g, respectively; and for fatty acids 34.76 ± 0.08 mg/g and 15.88 ± 0.06 mg/g, respectively. In conclusion, the SFE process at P 450 bar, T 60 °C and CS 53.33% of CO2 produced the highest yield of tocopherols, carotenoids and fatty acids. The MAE process at 400 W and 50 °C gives the best extracts in terms of tocopherols and carotenoids. For yield and fatty acids, the MAE process at 400 W and 70 °C produced the highest values. Both SFE and MAE showed to be suitable green extraction technologies for obtaining functional lipophilic compounds from Arthrospira platensis. Also, it is cheap, environmentally friendly and generally recognized as safe (GRAS) by the Food and Drug Administration (FDA) [2 has a high diffusivity with a tunable solvent strength [2 has been previously used for the extraction of functional lipophilic compounds from Arthrospira platensis such as carotenoids [Arthrospira only covers the extraction of protein pigments (C-phycocyanin) [Arthrospira by MAE.Microalgae and cyanobacteria cultivation has attracted the interest of industrial companies and the scientific community as a natural and sustainable biomass source for bioactive compounds ,2. Furthnditions ,4. Arthrorganism  considerorganism , includiorganism ,8. Commemoisture . Lipids moisture , besidesmoisture , antimicmoisture , and biomoisture . The maimoisture . Traditimoisture . In the on (FDA) . Furtherstrength . On the strength ,19. Addistrength . SFE witotenoids ,23, fattotenoids ,25 and totenoids ,27. Howeocyanin) , while focyanin) ,29 withoThe data provided below demonstrate the advantages of using green extraction technologies, since they are environmentally friendly and faster than traditional solvent extraction. Therefore, the goal of this study was to explore the effect of the extraction factors on the content of the functional lipophilic compounds in the extracts obtained by SFE and MAE, in order to determine the most important extraction factors and provide a framework for future work. In the present study, the tal runs ,31. The n = 2) and the last two experiments correspond to MAE (n = 3)) along with the extraction yields (as % w/w) showed as the mean ± standard deviation. For SFE, the highest extraction yield (4.07% ± 0.14% w/w) was achieved with experiment BDF, at 450 bar, 60 °C and 53.33% of co-solvent. When working with CO2 at supercritical conditions, the yield increases with the addition of the co-solvent, meaning that the extracted compounds are of different polarity and, therefore, the process becomes less selective [p < 0.05), where the highest temperature and highest amount of co-solvent gave the highest yield. In this response, pressure was not a significant factor (p > 0.05). For SFE, it has been reported that the main effect of a co-solvent is the solubility enhancement that results from an increase in solvent density and/or intermolecular interactions between the co-solvent and the solute [Arthrospira platensis extracts by SFE-CO2 [p < 0.05). Therefore, this shows that the use of higher temperatures in SFE and MAE led to the highest yields in these extraction processes. Moreover, the highest yield in MAE can also be attributed to the solvents used, as they have been widely reported as effective for obtaining lipophilic compounds with different polarities from different microalgae/cyanobacteria sources [SFE and MAE conditions were tested to cover a wide range of lipophilic compounds. elective . For yie SFE-CO2 , which c sources ,35.p < 0.05), while the temperature and co-solvent were not significant factors (p > 0.05). These results are similar to those obtained previously for Arthrospira species [p < 0.05). Furthermore, it was observed that a higher temperature  produced the lowest content. These results agree with various reports, stating that the most sensitive carotenoids (violaxanthin) in MAE usually start degrading at temperatures higher than 60 °C [et al. [Haematococcus pluvialis, where a high content of carotenoids was obtained. Also, our results agree with previous reports that suggest that efficient and fast extraction of carotenoids can be obtained using MAE without high pressure. Furthermore, the outstanding performance of MAE for carotenoids extraction could be attributed to the wall composition of A. platensis, since previous studies of pigment extractions report low efficiency of MAE with microalgae/cyanobacteria species that have a thick exopolysaccharide envelope [A. platensis. In the case of MAE, the highest content was observed at 50 °C. species ,36,37. Ian 60 °C . Zhao et [et al.  reportedenvelope . Our resenvelope ,39, showp < 0.05). The highest content of tocopherols was lower than previously reported [Arthrospira used in previous reports and in this study [p < 0.05), where the higher temperature negatively affected the content of tocopherols. This can be attributed to a possible degradation by heat [reported ; this mareported . Moreoveis study . For MAE by heat . Comparietc.; therefore, it is important to determine the fatty acid content in the extracts obtained with SFE and MAE. p < 0.05). The highest fatty acid content was found in treatments with 450 bar experiments ; as a consequence, pressure was an important parameter in the process of extraction of fatty acids by SFE. These results agree with previous reports of extraction of fatty acids with SFE. Machmudah et al. [Arthrospira, Sajilata et al. [et al. [Fatty acids represent one of the major building blocks of lipids . They cah et al.  reportedh et al. ,47. An ih et al. . In the h et al. . For fath et al. . The comh et al. ,48. For a et al.  reported [et al. .p < 0.05) was obtained with experiment H, 70 °C. This result is similar to those obtained by extraction yield at the same temperature. In the case of MAE, temperature is an important factor, since higher temperature produces the highest yield of compounds, although with the limitation of the thermal stability of the compounds present in the extract. According to the obtained results, the content of tocopherols and carotenoids decreased with increasing temperature. The composition of fatty acids in MAE extracts was found to be palmitic acid—52.64%, palmitoleic acid—3.72%, linoleic acid—15.47% and γ-linoleic acid—28.15%. Moreover, the content of γ-linolenic acid from MAE extracts was similar to the content of γ-linolenic acid from SFE extracts. However, there were differences in the content of palmitic acid, where MAE extracts (52.64%) were higher than SFE extracts (45.10%). In respect to linoleic acid, SFE extracts had higher content (21.27%) than MAE extracts (15.47%). This was due to differences in the affinity of the solvents used in the extraction process. This information is important since MAE offers higher purity extracts than other extraction technologies [For MAE the highest content of fatty acids (15.88 ± 0.06 mg/g)  were obtained from Tecnologia Ambiental Biomex S.A. de C.V. [Arthrospira was grown in open raceway ponds with modified Jourdan medium [Cyanobacteria samples  was used as co-solvent. An automated back pressure regulator (ABPR) controlled the extraction pressure, carbon dioxide pump was purchased from Thar Instruments  and the co-solvent pump from Waters .All extractions were carried out in a pilot-scale plant for supercritical fluid extraction  with a 100 mL extraction vessel (Waters Thar SFC SFE 100 Equipment). All extractions were carried out using a flow of 15 g/min of COg/g) was 37.5 in order to obtain the highest possible yield for SFE [2) (26.7 (E), 53.33 (F)). All extracts were concentrated in a vacuum rotary evaporator IKA Works  and kept under N2 at −20 °C in the dark. The selected response variables were extraction yield, concentration of carotenoids, concentration of tocopherols and concentration of fatty acids. The statistical method used for the data analysis was ANOVA, using the statistical package Minitab version 16 . All extractions were performed in duplicate.For each experiment, the extraction vessel was filled with 20 g of cyanobacteria powder (milled with mortar and pestle). The solvent/feed ratio  with a 100 mL extraction vessel GreenChem™. The extraction vessel was made of PFA Teflon™. All extractions were done using methanol/ethyl acetate/light petroleum (1:1:1 v/v) at 400 W power, 1 bar, and 15 min extraction time. For each experiment, the extraction vessel was filled with a 0.06 ratio of biomass/solvent (w/v). The effect of temperature was tested at two experimental levels T (°C) (50 (G), 70 (H)). All extracts were centrifuged at 4500 rpm, 4 °C for 7 min; the supernatants were transferred to evaporation flasks for concentration in a vacuum rotary evaporator  and kept under N2 at −20 °C in the dark; the residual pellets were discarded. The selected response variables were extraction yield, concentration of carotenoids, concentration of tocopherols and concentration of fatty acids. The statistical method used for the data analysis was ANOVA, using the statistical package Minitab version 16 . All Extractions were performed in triplicate.All extractions were carried out in a microwave-assisted extraction equipment MARS 5All extracts were made up to a final volume of 15 and 5 mL with ethyl acetate for SFE and MAE extracts, respectively. Total carotenoid content was estimated with UV-Vis spectrophotometer Hach DR5000  at absorbance maxima of 450 nm for β-carotene. Carotenoid content was calculated using an Tocopherols were analyzed with an Agilent 6890N GC  equipped with a HP-5MS capillary column  and a mass spectrometer 5973 N as a detector. The carrier gas was helium at a flow rate of 0.8 mL/min. The column temperature was kept initially at 190 °C for 1 min, then gradually increased to 300 °C at 15 °C/min, and finally, maintained at 300 °C for 10 min. For GC-MS detection, an electron ionization system was used with 70 eV of energy. An aliquot of the extracts (1 µL) was injected automatically with 20:1 in split mode. Injector and detector temperatures were set at 270 and 230 °C, respectively.v/v) and 2 mL of methanol/sulfuric acid (93:7 v/v). These mixtures were maintained for 60 min at 80 °C. Then, the samples were allowed to cool down to room temperature. After addition of 5 mL of hexane, the samples were mixed in a vortex for 1 min and two phases were formed. The organic phase was transferred to a volumetric flask (10 mL), and the re-extraction of the polar phase was carried out three to four more times until a volume of 10 mL was obtained. Fatty acids were determined through derivatization to fatty acid methyl esters (FAMEs). To prepare FAMEs of the extracts, samples (2 mL) were mixed with 400 μL of internal standard , equipped with a SP2380 capillary column  and flame ionization 19244–80560 as a detector. Nitrogen gas was used as carrier at a flow rate of 0.8 mL/min. The column temperature was kept initially at 50 °C for 2 min, then gradually increased to 240 °C at 4 °C/min, and finally, maintained at 240 °C for 1 min. An aliquot of the extracts (1 μL) was injected automatically with 20:1 in split mode. Injector and detector temperatures were set at 260 and 280 °C, respectively.Arthrospira platensis showed a significant amount of carotenoids, tocopherols and fatty acids compared to other cyanobacteria species. These extracts represent a sustainable and safe source of functional lipophilic compounds. In general, SFE extractions with ethanol produced higher amounts of these compounds as compared with MAE extractions. This represents an important advantage because ethanol is an economical and environmentally friendly solvent. However, for carotenoids, MAE was more effective than SFE. The SFE extraction (BDF) with 450 bar, 60 °C and 53.33% of co-solvent gives the best extracts in terms of yield, tocopherols, carotenoids and fatty acids. For MAE, the extraction at 400 W and 50 °C (G) gives the best extracts in terms of tocopherols and carotenoids. For yield and fatty acids, the best extracts were prepared at 400 W and 70 °C (H). SFE and MAE proved to be suitable green extraction procedures for functional lipophilic compounds from A. platensis.


To report our experience with small incision lenticule extraction ("SMILE") for myopia treatment.In this prospective clinical study, we evaluated 447 eyes from 224 patients with myopia, with and without astigmatism. We followed the patients for 6 months after SMILE.The mean  spherical equivalent was -6.75 ± 1.65 diopters (D) preoperatively and -0.21 ± 0.37 D at 6 months postoperatively. Our data showed that 97.9% of eyes were within ±1.0 D and 86.1% were within ±0.5 D of the intended correction. Furthermore, 79.8% had an uncorrected distance visual acuity (UDVA) of 0 logMAR or less  6 months after surgery. Additionally, 48.5% remained unchanged, 41% gained one line of corrected distance visual acuity (CDVA), 7.2% gained two lines of CDVA, 3.3% lost one line of CDVA, and 0.3% lost two or more lines of CDVA. Age was the only predictor for worsening UDVA at 6 months postoperatively in linear regression analyses . No predictor showed an association with error in spherical equivalent refraction at 6 months postoperatively.SMILE is an effective and safe refractive surgery. Age was the only predictor that influenced visual outcome, but its effect appeared clinically insignificant. Faster visual recovery is also expected with improved surgical technique. The procedure enables faster visual rehabilitation so patients can quickly return to normal activities[Laser-assisted tivities, 2. Howetivities, ectasiativities, and trativities. Recentltivities. It has tivities, 8. Additivities.In the current study, we investigated the efficacy, predictability, and safety of SMILE and compared our results with those from previous studies, 10–14. The Institutional Review Board/Ethics Committee of Yeouido St. Mary’s Hospital approved this study (SC14RISI0036). It was performed in accordance with the tenets of the Declaration of Helsinki. Written informed consent for participation in the study was obtained from all participants. Inclusion criteria for the study were as follows: spherical myopia up to -12.0 D, myopic astigmatism up to -4.0 D cyl, minimum age of 18 years, corrected distance visual acuity (CDVA) of 20/40 or better (>0.3 logMAR), and minimum calculated postoperative residual stromal bed of 250 μm. In addition, patients had no ocular conditions other than myopia. From May 2012 to November 2013, 447 eyes from 224 patients with myopia (with and without astigmatism) who were treated consecutively with SMILE at the Onnuri Eye Clinic, Jeonju, Korea, fulfilled these criteria. Patients were followed for 6 months.Patients underwent preoperative examinations including autokeratometry, autorefractometry, intraocular pressure tonometry , pupillometry , corneal tomography, corneal thickness measurements , measurement of uncorrected (UDVA) and corrected distance visual acuity (CDVA), measurement of manifest and cycloplegic clinical refraction, slit lamp evaluation, and fundoscopy.A VisuMax 500-kHz femtosecond laser  was used for SMILE treatment. The surgery was performed bilaterally and under topical anesthesia using three drops of 0.8% oxybuprocain tetrachloride 2–3 min before surgery. The patient was positioned under the curved contact glass of a femtosecond laser and was asked to fixate on a blinking target. Once appropriate centration was achieved, suction was applied to the contact glass. We used 500 kHz, cut energy index 180 nJ femtosecond laser pulsed, and 4.5-μm spot spacing. First, the back of the intrastromal lenticule was created by photodisruption from the periphery to the center, followed by creation of the lenticule front from the center to the periphery and an incision tunnel located at 11 o/c. The lenticule diameter was 6.5 mm and the cap diameter was 7.5 mm. The incision length was 2.0 to 2.5 mm. The intended cap thickness was 100 to 120 μm. After laser treatment, a thin blunt spatula was inserted through the incision site to break the remaining tissue bridges and loosen the stromal lenticule, which was pulled out using McPherson forceps  and removed. After the removal of the lenticule, the stromal pocket was flushed with saline. After surgery, all patients received a topical antibiotic for 5 days and a topical steroid for 2 weeks. Hyaluronic acid lubricating drops were prescribed for at least 2 weeks.The follow-up appointments occurred 1 day, 1 week, 1 month, 3 months, and 6 months after surgery. At each visit, CDVA, UDVA, objected and manifest refractions, keratometry, intraocular pressure, corneal topography, and slit lamp examination were performed. Complications were noted.2 analyses were used to compare success rates of early postoperative period visual recovery among groups with different incision lengths. P values less than 0.05 were considered to indicate statistical significance.Statistical analyses were performed using the SPSS software . Graphics were generated using Microsoft Excel 2007 . All values are given as the means ± standard deviation. Statistical analyses for visual acuity are based on logMAR units. Bivariate correlation analyses and multiple linear regression analyses were used to evaluate prognostic factors for visual outcomes. The preoperative patients and surgical-related parameters included age, gender, eye side (right/left), corneal curvature, corneal thickness, intraocular pressure, attempted change in spherical equivalent refraction, and degree of astigmatism. Pearson’s χThis study included 293 eyes from 147 females (65.5%) and 154 eyes from 77 males (34.5%). The mean age was 27.05 ± 6.10 years . Table 2 test). However, there was no statistically significant difference among the groups at 1 week after surgery .Figure Figure t-test). Safety is presented in Figure Postoperative SE values were -0.18 ± 0.4 D after 1 week, -0.20 ± 0.39D after 1 month, -0.21 ± 0.38D after 3 months, and -0.21 ± 0.37D after 6 months  to precisely evaluate the predictability, efficacy, and stability of SMILE and to determine predictors that influence visual outcome.The efficacy, predictability, and safety of SMILE in the present study were promising and comparable with other recently published studies–12. RegaFor predictability, 80.1%, 77%, 91% and 92% were within ±0.5 D and 94.2%, 95%, 98% and 100% were within ±1.0D of the attempted refraction in the various previous studies. In the Regarding safety, Hjortdal et al. reportedIn the present study, 0.3% of eyes lost two lines, 3% lost one line, and 96.7% were the same or improved at 6 months after surgery. Anders et al. reportedAccording to Hjortdal et al., age, coThe slight delay in UDVA recovery in the early postoperative period is a unique characteristic of FLEx and SMILE–16. Shah11–12, 81.2% of patients had similar UDVA values in the present study. In particular, 91.4% and 96.6% of patients who received 2.0 mm incision lengths (n = 58) had UDVA 20/25 or better on the first day and 1 week after surgery, respectively.We found that the initial visual recovery was faster compared with recent studies that also used the newer-generation femtosecond laser (500 kHz). Although 40% and 62.3% of patients had UDVA that was 20/25 or better on the first postoperative day in previous reportsThe faster initial visual recovery noted in the present study may be attributed to the use of different laser settings or to improved surgical factors, such as smaller incision lengths. Laser settings of higher pulse energy and larger spot spacing appear to aid faster initial visual recovery. The laser setting of 180-nJ pulse energy and 4.5-μm spot spacing in the present study showed faster initial visual recovery compared to the previously reported 120 to 150-nJ pulse energy and 2.5-μm spot spacing. AdditioTo our knowledge, no reported study has evaluated the effect of incision length on visual outcome after SMILE. The present study found that as incision length decreased, visual recovery on the first postoperative day was more rapid (P = 0.013). However, there were no significant differences among the various incision length groups after 1 week. The length of incision did not affect other refractive results, including error in SE and amount of astigmatism after surgery. However, there are several weaknesses in the comparisons among incision groups. Although all eyes were treated by one surgeon, there was no randomization of the incision groups and the numbers of eyes differed significantly by group. Moreover, this analysis of the effects of incision size was, in fact, retrospective, because we had not planned to analyze the effects of incision size on visual outcome when the study was designed. There is also a possibility that the surgeon may have become more skillful when the smaller incision group was treated compared with when the larger incision group was treated, in a relatively early period. In our experience, as the surgeon became adept at smaller incisions, the surgeon could achieve manipulations more readily, as with a larger incision, during the SMILE procedure. Thus, it becomes possible to benefit from the smaller incision, with increased corneal stability and rapid epithelial healing, which may aid in more rapid visual recovery. Presently, we cannot exclude the possibility of better initial visual outcomes because of the smaller incisions, not only greater surgeon experience. The relationship between incision length and HOA after surgery should be evaluated in future studies.Hjortdal et al. reportedOne eye that had undercorrected SE of -1.63 D and 20/40 UDVA after 6 months was retreated in the present study. The eye had a refraction of emmetropia (-0.25 D) and 20/20 UDVA with no other complication 3 months after the surface ablation procedure (PRK).The present study had a large number of samples, relatively long-term observation, and treated moderate-to-severe myopia. Our data indicate that SMILE is an effective and safe refractive surgery. Moreover, by developing laser platforms and improving surgical techniques, it is anticipated that the slow visual recovery that is considered a drawback of SMILE can be improved. This procedure would likely be preferred by patients who desire safer refractive surgery with faster visual recovery.


Toxoplasma gondii is a zoonotic pathogen that can infect a range of animals and humans. Ingestion of T. gondii oocysts in soil is a significant transmission route for humans and animals acquiring toxoplasmosis. In the present study, we developed a new semi-nested PCR method to determine T. gondii oocysts distribution in soils in northwestern China.T. gondii in soil, targeting the repetitive 529 bp fragment of T. gondii genomic DNA. Then a total of 268 soil samples, including 148 samples from Gansu Province and 120 samples from Qinghai Province, northwestern China, were examined by the semi-nested PCR method. One third of the positive samples were sequenced. The sensitivity of the semi-nested PCR assay was 102 T. gondii oocysts in 5 g soil sample. Investigation of soil samples from northwestern China showed that 34 out of 268 soil samples (12.69%) were T. gondii positive. Sequences of the partial 529 bp fragments varied from 0–1.2% among the sequenced samples. The prevalence of T. gondii oocysts in soil from cities (24/163) was slightly higher than that in soils from pasturing areas (10/105) (P = 0.21). Among the different regions in cities, the prevalence of T. gondii oocysts in soils from parks was 14.15%, whereas that in soils from schools was 19.05%.The one tube semi-nested PCR assay was developed to detect the oocysts of T. gondii oocysts in soils in northwest China using a novel semi-nested PCR assay, which provided baseline data for the effective prevention and control of toxoplasmosis in this region.The present study firstly reported the prevalence of  Toxoplasma gondii is a zoonotic infection of human beings and animals [T. gondii infection in livestock usually causes abortions, stillbirths, and neonatal deaths, especially in sheep and goats, which have been leading to serious economic losses worldwide [Toxoplasmosis caused by  animals -5. The p animals ,6,7. T. orldwide ,4.T. gondii are resistant to harsh climatic circumstances [T. gondii oocysts after shedding from felids is considered significant routes of T. gondii transmission to humans and animals [The sporulated oocysts of mstances ,4. The m animals . Infecti animals ,9-11. Th animals -14. The  animals ,16.T. gondii, previous studies developed several methods such as loop-mediated isothermal amplification (LAMP) and QT-PCR to investigate its oocysts in soils [T. gondii oocysts prevalence in soils could be available in northwestern China, especially in the pasturing areas. The objective of the present study was to investigate prevalence of T. gondii oocysts in the soil samples from Qinghai and Gansu provinces in northwest China. Due to the low concentration of T. gondii contained in soils, we developed a new semi-PCR method based on the 529-bp repeat element to improve the sensitivity.Due to the important role of contamination of soil in the transmission dynamics of in soils -20. HoweThis study was approved by the Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences , and the soil samples were collected strictly according to the requirements of the Ethics Procedures and Guidelines of the People’s Republic of China.The present study was carried out in Qinghai province  and Gansu province , which lie on the northwestern of People’s Republic of China. The average elevation of Qinghai province is 3 000 m above sea level, and Gansu province is more than 1 000 m above sea level. The annual precipitation of the survey regions are below 500 mm and average annual temperature are between −4.3°C and 14.8°C. The survey regions belong to typical continental climate.148 soil samples from 5 schools and 2 parks were collected in Lanzhou city, and the other 120 samples were collected from Huzhu, Ping’an, Huangyuan, Xining, Haibei and Hainan cities in Qinghai Province. Thereinto, Xining and Lanzhou cities are capitals of Qinghai and Gansu Provinces, respectively, and the rest 5 cities locate in the pasturing areas.T. gondii GJS strain orally via a stomach tube. One week after the challenge, the T. gondii oocysts were isolated from feces and were purified using differential centrifugation according to Haydee et al. [Three captive-bred cats (three-month old) were inoculated e et al. . The sedT. gondii oocysts, the specific semi-nested PCR reaction was developed targeting the T. gondii 529-bp fragment (accession number: AF146527). The length of targeting fragment to design the primers was 344 bp, with the primers of TX1: 5′-CAGGGAGGAAGACGAAAGTTG-3′, TX2: 5′-CACAGAAGGGACAGAAGT-3′, TX3: 5′-CTGTGTCACGTAGACCTAAGG-3′. The amplification mixture consisted of 12.5 μl of 2 × reaction mix , 0.004 μM of TX1, 0.2 μM of TX2, 0.2 μM of TX3, 0.5 μl of 1% BSA, and 1 μl of soil template DNA in a final volume of 25 μl. The reaction mixture was initially incubated for 3 min at 95°C to denature the template DNA, and then samples were amplified as follows: 15 cycles of denaturation at 95°C for 30 s and annealing/extension at 64°C for 40 s, extension at 72°C for 5 min, 35 cycles of denaturation at 95°C for 30 s and annealing/extension at 55°C for 40 s, extension at 72°C for 8 min. Semi-nested PCR product was subjected to electrophoresis on a 2.0% agarose gel in a Tris-acetic acid-EDTA (TAE) buffer at 80 V for 30 min and visualized under UV light after staining with ethidium bromide.To detect 6, 105, 104, 103, 102, 101, and 100. Every soil sample  was filtered by double gauzes, and then genomic DNA of T. gondii from soil samples were extracted from 0.4 g soil using the soil DNA kit  according to the manufacturer’s instructions. The DNA was eluted in 70 μl elution buffer and stored at −20°C until further analysis.Seven oocyst-free soil samples (5 g) were prepared in a laboratory and experimentally contaminated with oocysts in the following amounts: 10T. gondii oocysts were isolated as described above. The samples were subjected to the developed semi-nested PCR to detect T. gondii oocysts in soil. Each sample was performed in triplicate. Ten of the positive samples were randomly collected and then sent to Sangon Biotech Co. Ltd. (China) for sequencing.To evaluate the feasibility of the semi-nested PCR assay for the analysis of field samples, a total of 268 field soil samples were collected from Gansu and Qinghai provinces, China. The genomic DNA of T. gondii between regions in soil samples were analyzed by Chi-square analysis in SAS . Values of P < 0.05 were considered as statistically significant.The difference in the prevalent rates of 2 T. gondii oocysts in 5 g soil sample  and 3 transversions (A < − > T and C < − > G). The prevalence of T. gondii oocysts in soils from cities (24/163) was higher than that from pasturing areas (10/105), and the difference was not statistically significant (P = 0.21). Among the different regions in Lanzhou city, the prevalence in soils from schools was 14.15% (15/106), which was statistically not significantly different to that in soils from parks   soil samples. The prevalence of oocysts in soil was lower than that in Hubei province, China by PCR method and in São Paulo, Brazil by immunohistochemistry and indirect fluorescent antibody test (IFAT) [T. gondii oocysts, and were unfavorable for epidemics of toxoplasmosis [A total of 268 soil samples from northwestern China were further applied in epidemiological studies with the semi-nested PCR method. Positive DNA against t (IFAT) ,22. The lasmosis ,26, whiclasmosis  and poullasmosis  in Lanzhlasmosis  was loweT. gondii oocysts . The widespread distribution of T. gondii in city was mainly caused by stray or free-living cats. In our previous study, we isolated 11 T. gondii strains from 41 stray cats [T. gondii are contributed to the high prevalence of the parasite and constant infection pressure in the environment [City was >1 × more than pasturing area to be contaminated ray cats , therefoironment . We can T. gondii prevalence in schools indicates a main risk source of infection for younger, especially the presence in soil from school C and E. The school C and E were elementary schools. Students, sometimes, may play with earth. They may be infected with T. gondii due to blot hands with the oocysts contaminated soil and then eat food without any awareness. So, parents should be take appropriate precautions. In addition, regularly cleaning the surroundings and other integrated strategies and measures are necessary for the effective prevention and control of T. gondii oocysts prevalence.The T. gondii genomic DNA. Using this assay, high level (12.69%) contamination of T. gondii oocysts in soils in northwestern China was revealed for the first time, indicating a potential risk of soil as an important source of T. gondii infection in humans and other animals in this region of China.The present study developed a new semi-nested PCR assay based on the repetitive 529 bp fragment of


In total, 7 adverse drug reactions occurred in 4 subjects during the study; of these, 3 cases were from 3 subjects in the test treatment group, and 4 cases were from 3 subjects in the reference treatment group. All adverse drug reactions had been reported previously, and all subjects recovered fully without any sequelae. In conclusion, the pharmacokinetic profiles of metformin in two different study treatments, a voglibose/metformin FDC vs. the coadministration of the individual formulations, met the regulatory criteria for bioequivalence in healthy Korean subjects under fasting conditions. There was no significant difference in safety profiles between the two treatments.  The aim of this study was to compare the pharmacokinetic characteristics of metformin between a fixed-dose combination (FDC) of voglibose/metformin and co-administered individual voglibose and metformin tablets in healthy Korean volunteers under fasting conditions. A randomized, open-label, single-dose, two-treatment, two-way crossover study with a 7-day wash-out period was conducted. Plasma samples were collected for up to 24 hours and were analyzed for metformin using a validated liquid chromatography tandem mass-spectrometry (LC/MS). A non-compartmental method was used to calculate the pharmacokinetic parameters. Vital signs and adverse events were monitored, and physical examinations and laboratory tests were conducted to evaluate safety. In total, 28 subjects completed the study. The geometric mean ratio (GMR) and the 90% confidence interval (CIs) of C Clinical Trial Registrationhttp://clinicaltrial.gov/ClinicalTrials.gov number, NCT01370681. Available at URL. Diabetes mellitus (DM) is a disease of metabolic dysregulation, characterized by chronic hyperglycemia due to insufficient insulin action , 2. The Metformin, an oral antihyperglycemic agent, is extensively used in the treatment of type 2 diabetes. Metformin reduces gluconeogenesis in the liver by activating AMP-activated protein kinase (AMPK) via liver kinase B1 (LKB1) . It alsoVoglibose, an α-glucosidase inhibitor, is also used widely in the management of type 2 diabetes. It undergoes minimal systemic absorption . VogliboAccording to the American Diabetes Association guideline, metformin is suggested as the preferred initial agent, and, if metformin monotherapy fails to reduce or maintain blood glucose, other therapeutic agents should be added to metformin . Fixed-dThe objective of this study was to evaluate the bioequivalence of metformin administered as a single dose of a voglibose/metformin 0.2/500 mg FDC tablet vs. the co-administration of voglibose 0.2 mg and metformin 500 mg in healthy Korean volunteers under fasting conditions. Only the pharmacokinetics of metformin were evaluated in this study because voglibose is undetectable after therapeutic dosing . Healthy volunteers, aged 20 – 55 years, who were within 20% of their standard body weight according to the Broca formula, were considered for participation. Subjects who had not experienced congenital or chronic disease, were judged to be healthy based on the results of a detailed clinical examination, could participate in the whole clinical trial, and voluntarily signed a written informed consent form were eligible for inclusion in this study. The following exclusion criteria were used: administration of inducers or inhibitors of drug-metabolizing enzymes within 1 month, symptoms of acute disease within 4 weeks, a history of allergic disease or hypersensitivity reactions, abnormal laboratory test results, excessive consumption of caffeine (> 5 cups/day), cigarettes (> 10 cigarettes/day), or alcohol , a diet containing foods known to affect the absorption, distribution, metabolism, and excretion of the study drugs , a history of participation in another clinical study within 90 days, a whole-blood donation within 60 days, pregnant/lactating females, and females of childbearing potential not practicing a medically acceptable method of contraception. The study protocol was approved by the Inje University Busan Paik Hospital Institutional Review Board (IRB No: 09-117). The study was carried out in compliance with the Declaration of Helsinki, the International Conference on Harmonisation of Good Clinical Practice (ICH-GCP), and the current Korean Good Clinical Practice (KGCP) guidelines. The clinical trial was conducted at the Inje University Busan Paik Hospital clinical trial center, Busan, Korea, from October 29, 2009 to November 17, 2009 . All participants were informed of the study objectives, potential risks, and compensation before joining the study. All eligible subjects provided written informed consent to participating and were free to withdraw from the study at any time without obligation. Voglibose/metformin 0.2/500 mg FDC  was used as the test treatment, and the coadministration of voglibose 0.2 mg  and metformin 500 mg  were used as the reference treatment. This was an open-label, randomized, single-dose, two-way crossover study in healthy male subjects. Subjects were allocated to each group  in a 1 : 1 ratio according to a predesigned randomization table that was generated using SAS software . There was a screening period of up to 28 days prior to study drug administration on day 1. Subjects received one of the two treatments according to the group they were allocated to. After a 7-day wash-out period, which was determined to be more than seven times the half-life of metformin  and convAfter an overnight fast prior to study drug administration on day 1, the study drug was administered with 240 mL tap water, supervised by physicians. An oral check was performed immediately after the administration in each subject to ensure compliance. Water was permitted 2 hours after the study drug administration and food 4 hours after. For the pharmacokinetic analysis of metformin, blood samples were collected at pre-dose (0 hour) and 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 8, 10, 12, and 24 hours after administration in each period. Whole blood samples (7 mL) were collected in heparinized vacutainer tubes, and then centrifuged  to separate plasma. Plasma samples (1 mL) were transferred to microcentrifuge tubes and stored at –80 °C until analysis. Subjects who received at least one treatment during the study were included in the safety assessment analysis. To evaluate safety, vital signs , physical examination, and laboratory tests  were assessed. Vital signs were measured before (0 hours), and at 3, 12, and 24 hours after administration in each period. Physical examination and laboratory tests were evaluated at the time of hospitalization and discharge. Metformin concentrations in plasma were analyzed using liquid chromatography tandem mass spectrometry , based on a method developed previously . Metform2) greater than 0.994. The intra- and interday precision was 1.70 – 5.31 and 4.44 – 11.33%, respectively. The intra- and interday accuracy was 97.31 – 112.01% and 100.94 – 106.95%, respectively. There was no significant interference in selectivity or stability. Briefly, 500 µL of acetonitrile containing the internal standard  were added to 100 µL of plasma sample. After vortex-mixing for 10 minutes and centrifugation , the supernatant was injected into the LC-MS/MS system. A full validation of the assay was carried out with respect to selectivity, accuracy, precision, calibration curve, and stability. The calibration curve of metformin was linear over the range of 10 – 2,000 ng/mL, and the lowest limit of quantification was 10 ng/mL, with a coefficient of determination  recommends that bioequivalence studies should be conducted with at least 12 subjects per group  . 30 subjetformin , was calmax) and area under the concentration-time curve from zero (predose) to time of last quantifiable concentration (AUC0–t) were the primary parameters, and time to reach Cmax (tmax), half-life (t1/2), and AUC0–∞ were estimated as secondary parameters. Cmax and tmax were obtained from the observed plasma concentration-time profile. AUC0–t was calculated according to the linear trapezoidal rule. AUC0–∞ was calculated with the following equation: AUC0–∞ = AUC0–t + Ct/λz, where Ct is the last observed concentration and λz is the elimination rate constant. The t1/2 was calculated as 0.693/λz. Subjects who completed the study were included in the pharmacokinetic analysis. The pharmacokinetic parameters of metformin were estimated using a noncompartmental method with WinNonlin software . The maximum concentration  of the geometric mean ratio of primary parameters were within the range 0.8 to 1.25, the new formulation was considered to meet the criteria for bioequivalence [max and AUC with random effects of sequence-nested subject, and fixed effects of sequence, period, and treatment. All statistical analyses were performed using the SAS software . A p-value of < 0.05 was considered to indicate statistical significance. For the bioequivalence test, the geometric mean ratios of Civalence , 27. A mIn total, 30 healthy subjects were enrolled. Two subjects dropped out before the first administration of the investigational product because of laboratory test abnormalities. Thus, 28 subjects participated from the beginning of the study and completed the study. They were included in the pharmacokinetic analysis and safety assessment. Their mean  age, height, and weight were 23.97 (1.56) years, 174.67 (6.10) cm, and 72.03 (8.27) kg, respectively. max values of metformin after administration of the test and reference treatments were 1.38 (0.32) and 1.35 (0.30) µg/mL, respectively. The mean (SD) AUC0–t and AUC0–∞ values were 8.17 (1.65) and 8.28 (1.65) µg×h/mL, respectively, after administration of the test treatment. The mean (SD) AUC0–t and AUC0–∞ values were 7.63 (1.54) and 7.73 (1.54) µg×h/mL, respectively, after administration of the reference treatment , funded by the Ministry of Health & Welfare, Republic of Korea (HI14C1063). We thank Su-Jin Park and Ji-Eun Seo for their support in this clinical trial. Eun Ji Kim and Geun Seog Song are full-time employees of Clinical Development Division, CJ HealthCare Corp. The other authors have no conflicts of interest to disclose.


Bacillus cereus IND1 was isolated from the Indian food, rice. Solid-state fermentation was carried out using agroresidues for the production of fibrinolytic enzyme. Among the substrates, wheat bran supported more enzyme production and has been used for the optimized enzyme production by statistical approach. Two-level full-factorial design demonstrated that moisture, supplementation of beef extract, and sodium dihydrogen phosphate have significantly influenced enzyme production (P < 0.05). A central composite design resulted in the production of 3699 U/mL of enzyme in the presence of 0.3% (w/w) beef extract and 0.05% (w/w) sodium dihydrogen phosphate, at 100% (v/w) moisture after 72 h of fermentation. The enzyme production increased fourfold compared to the original medium. This enzyme was purified to homogeneity by ammonium sulfate precipitation, diethylaminoethyl-cellulose ion-exchange chromatography, Sephadex G-75 gel filtration chromatography, and casein-agarose affinity chromatography and had an apparent molecular mass of 29.5 kDa. The optimum pH and temperature for the activity of fibrinolytic enzyme were found to be 8.0 and 60°C, respectively. This enzyme was highly stable at wide pH range (7.0–9.0) and showed 27% ± 6% enzyme activity after initial denaturation at 60°C for 1 h. In vitro assays revealed that the enzyme could activate plasminogen and significantly degraded the fibrin net of blood clot, which suggests its potential as an effective thrombolytic agent.A potent fibrinolytic enzyme-producing  Bacillus natto, had potent thrombolytic activity. These bacterial fibrinolytic enzymes are generally safe, and the oral administration of the nattokinase enzyme could increase the fibrinolytic activity in human plasma [ natto, tofuyo, Korean cheonggukjang soy sauce, edible honey mushroom [ douche [ tempeh [Cardiovascular diseases (CVDs) including acute myocardial infarction, ischemic heart disease, peripheral vascular disease, high blood pressure, and stroke are the leading causes of death worldwide . Accordin plasma . Fibrinomushroom , Chinese[ douche , Indones[ tempeh , Taiwane[ tempeh , Japanes[ tempeh , and Asi[ tempeh . These f[ tempeh . Fusarium oxysporum [ Bacillus firmus NA-1 and soybean grits [α-amylase [β-galactosidase [Solid-state fermentation (SSF) has emerged as a potential technology for the production of pharmaceutically significant products. Many agroindustrial residues were used for the production of these products using SSF. The agroindustrial residues such as pigeon pea, green gram husk, potato peel, and wheat bran were widely used for the production of proteases –14. Utilxysporum  and Bacian grits ). Responan grits , argininan grits , α-amyla-amylase , acid pr-amylase , and β-gtosidase . Howevertosidase . in vitro.The aim of this work was to optimize the fermentation medium by RSM to increase fibrinolytic enzyme production in SSF. The optimization procedure included the following: a two-level full-factorial design, a central composite design (CCD), and an RSM. The extracted fibrinolytic enzyme was purified by chromatographic methods and was used to study its blood clot lytic properties Bacillus cereus and incubated at 37°C for 72 h. The pH of the fermenting medium was maintained as 8.0 using Tris buffer (0.1 M) and 100% (v/w) moisture. Fibrinolytic activity of the crude enzyme was tested in a fibrin plate composed of 0.1 M sodium phosphate buffer (pH 7.4), 1% (w/v) agarose, 1.2% (v/v) fibrinogen, and thrombin (100 NIH units/mL) [μL of crude enzyme was dropped into holes and incubated at 37°C for 5 h. The fibrinolytic enzyme exhibited a clear zone of degradation of fibrin around the well, thus indicating its fibrinolytic activity.Approximately 1.0 g of fermented rice was transferred to an Erlenmeyer flask (250 mL) with 50 mL of sterile double-distilled water, shaken vigorously for 10 min, and 1 mL of this solution was resuspended in sterile double-distilled water and aliquots were then spread on nutrient agar plates composed of the following (g/L): peptic digest of animal tissue, 5.0; beef extract, 1.5; yeast extract, 1.5; sodium chloride, 5.0; and 10 skimmed milk (pH 7.0). Colonies, indicated by clear zones corresponding to protease activity, were selected and cultured in SSF using wheat bran as the substrate. SSF was performed by inoculating 10% (v/w) preculturednits/mL) . The fib B. cereus IND1. The 910 bp sequences were submitted to the GenBank database, and an accession number was assigned to those sequences. The GenBank accession number of the sequence reported in this paper is KF250417.The isolated strain IND1 with highest activity was identified on the basis of the biochemical properties, the phenotypical characteristics , and theThe substrates such as banana peel, tapioca peel, rice bran, wheat bran, and green gram husk were collected locally and dried for several days and powdered. About 2.0 g of substrates was taken in an Erlenmeyer flask, and the moisture content was maintained as 100% level. The contents were mixed thoroughly and inoculated with 0.2 mL of 18 h grown culture (OD 600 nm = 1.08). To the fermented medium, 20 mL of double-distilled water was added and placed in an orbital shaker at 150 rpm for 30 min. After this, it was centrifuged at 10,000 ×g for 10 min, and the supernatant was used as the crude enzyme.2, pH 7.8), and 0.05 mL of enzyme solution. After 30 min at 37°C, the reaction was stopped by adding 5 mL of 0.11 M trichloroacetic acid containing 0.22 M sodium acetate and 0.33 M acetic acid. The reaction mixture was centrifuged at 10,000 ×g for 10 min, and the absorbency of the sample was read at 275 nm against a sample blank. A fibrinolytic unit was defined as the amount of enzyme that gave an increase in absorbency at 275 nm equivalent to 1 μg of tyrosine/min at 37°C. The total protein content determination was performed as described by Lowry et al. [Fibrinolytic activity of the sample was measured by the hydrolysis of fibrin . The rea5 factorial design and the RSM. With the results obtained from the one-at-a-time strategy, three nutrients were selected for further optimization. Important nutrient factors such as maltose, beef extract, and NaH2PO4 were added to the solid substrate according to 25 full-factorial design. From an SSF point of view, moisture content is one of the significant factors. Hence, the optimum moisture content and pH were evaluated with respect to the production of the fibrinolytic enzyme. Based on the two-level full-factorial design, each factor was examined at two levels: −1 for low level and +1 for high level. Y  regarding the fibrinolytic enzyme production. Each of the variables used was analyzed at five coded levels  as listed in 5 factorial design). About 2.0 g of wheat bran was taken in an Erlenmeyer flask and mixed with a predetermined quantity of Tris buffer , and a calculated amount of beef extract and NaH2PO4 was also added to the flask. The substrate and nutrients were mixed carefully and sterilized at 121°C for 20 min. All the Erlenmeyer flasks were inoculated with a 10% (v/w) inoculum and incubated at 37°C for 72 h. Twenty milliliters of sterilized double-distilled water was added to extract the fibrinolytic enzyme unless otherwise stated. The enzyme activity assay was carried out in duplicates, and the average of these experimental values was taken as response Y ; A is the coded value of moisture; B is the coded value of the beef extract; C is the coded value of NaH2PO4; α1, α2, and α3 are the linear coefficients; α1α2, α1α3, and α2α3 are the interactive coefficients; and α1α1, α2α2, and α3α3 are the quadratic coefficients.Consider the following:Response surface graphs were plotted to determine the optimum fibrinolytic enzyme production. The fitted polynomial equation was expressed as three-dimensional (3D) surface plots to visualize the relation between responses and the experimental levels of each factor used in the design. Validation of the model was performed under the conditions predicted by the model. SSF was carried out, and fibrinolytic activity was assayed as described earlier.The crude extract was precipitated by the addition of solid ammonium sulfate at 30%–70% saturation. The precipitate was allowed to form at 4°C overnight and was collected by centrifugation at 10,000 ×g in a refrigerated centrifuge for 15 min. The precipitate was dissolved in 5 mL of buffer A . It was dialysed against buffer A overnight and loaded on DEAE-cellulose  column, which was preequilibrated with buffer A. The column was washed with buffer A to remove all unbound proteins, and a linear gradient of 0–0.75 M NaCl-added buffer A was used to elute the bound proteins. Fractions exhibiting fibrinolytic activity were pooled and concentrated with ammonium sulfate. The precipitate was collected by centrifugation at 10,000 ×g in a refrigerated centrifuge at 4°C for 15 min, dissolved in buffer A, and dialysed against the same buffer overnight. This sample was loaded on Sephadex G-75  gel filtration column (0.7 × 45 cm) which was preequilibrated with buffer A. The eluate was monitored for protein concentration at 280 nm and was assayed for fibrinolytic activity. Fractions with high fibrinolytic enzyme activity were pooled and loaded on casein-agarose affinity column (Sigma) and washed with buffer A. The fibrinolytic enzyme was eluted with buffer A containing 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, and 0.8 M NaCl. All fractions were subjected to fibrinolytic activity assay. The purified enzyme was stored at −20°C and used for characterization studies.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis  was performed to determine the molecular mass of the fibrinolytic enzyme following the methods of Laemmli . The mol2+, Co2+, Cu2+, Mg2+, Mn2+, Hg2+, Fe2+, and Zn2+. The enzyme activity was determined as described earlier.The optimum pH for the activity of an enzyme was determined using the following buffers (0.1 M): citrate buffer (pH 3.0 and 4.0), succinate buffer (pH 5.0), sodium phosphate buffer (pH 6.0 and 7.0), Tris buffer (pH 8.0), and glycine-NaOH buffer (pH 9.0 and 10.0). The stability of fibrinolytic enzyme activity in response to pH was evaluated by incubating the enzyme with the above buffers at 37°C for 1 h prior to incubation with substrate. The effect of temperature on enzyme activity was determined by performing the reactions at various temperatures: 30, 40, 50, 60, and 70°C. To determine the thermal stability, the fibrinolytic enzyme was incubated  for 1 h. Enzyme activity was assayed as described earlier. To study the effect of divalent ions on enzyme activity, the enzyme sample was incubated with various divalent ions, namely, CaThe plasminogen-rich and plasminogen-free plates were prepared to evaluate the efficacy of fibrinolytic enzyme on plasminogen and direct fibrin clot lysis. The fibrin plate was heated at 80°C for 30 min to inactivate plasminogen present with the commercially available fibrinogen. Twenty microliters of fibrinolytic enzyme was placed on the plasminogen-free and plasminogen-rich plates and incubated at room temperature for 4 h. The enzyme exhibited a clear zone of degradation of fibrin on plasminogen-rich plate and plasminogen-free plate was observed. in vitro. The blood was collected from healthy male volunteer with written informed consents. Artificial blood clot was made by spontaneous coagulation in the centrifuge vials. Then, different doses  of the fibrinolytic enzyme were added. Streptokinase (250 units) was used as the positive control; buffered saline solution was used as the negative control. These were incubated at room temperature for 60 min and analyzed.The clot lytic effect of fibrinolytic enzyme was studied with an artificial clot B. cereus IND1 displayed more activity on skimmed milk agar plates and fibrin plates. It produced approximately an 11 mm zone on the fibrin plate, which was higher than the other isolates. The isolated strain was Gram positive, rod shaped, catalase positive, and oxidase positive and had tested negative for indole formation, citrate utilization, and the hydrolysis of urea. It was able to hydrolyze starch and casein and also tested negative for gelatin hydrolysis.The bacterium B. cereus IND1 varied with the type of substrates. Enzyme production was found to be high in the wheat bran medium  is significant at the 5% level. There is only a 0.80% chance that a “model F-value” this large could occur due to noise. In this model, B, C, E, AC, BC, CD, ABD, ADE, BDE, CDE, ABCE, and BCDE are significant. Maltose significantly increased the fibrinolytic enzyme production. These results were in accordance with reported fibrinolytic enzyme production in the presence of maltose for Bacillus natto [2PO4 concentration affected the fibrinolytic enzyme production significantly. Enzyme production was greatly affected by moisture (P < 0.01), beef extract (P < 0.01), and NaH2PO4 (P < 0.05) concentrations with the medium. The equation in terms of the coded factors is given as follows:The 2us natto . The medt values, the moisture content, beef extract, and NaH2PO4 were selected for further optimization using RSM.On the basis of calculated 2PO4) were investigated further using CCD and RSM. The CCD model helps to study the interactions between the various variables, and RSM helps to explore the optimum concentrations of each of the variables. RSM had been successfully used for the enhancement of fibrinolytic enzyme production by the Bacillus natto [ Bacillus subtilis [ Bacillus sp. strain AS-S20-1 [RSM is a powerful technique for testing multiple process variables, because fewer experimental trials are required when compared with the study of one variable at a time. Also, interactions between variables can be identified and quantified by this technique . The sigus natto , Bacillusubtilis , and BacAS-S20-1 .F-value of 9.76 implied that the model was significant  and the quadratic model term (A2) were significant. However, the interactive model terms  and quadratic model terms (B2 and C2) were found to be insignificant (P > 0.05). Yuguo et al. [R2) could be at least 0.8 for a good fit of the model. The multiple correlation coefficient (R2) of this model is 0.89 , which means that the model can explain 89% of the variation in the response. The adjusted coefficient (R2) obtained for the model was 0.805. According to ANOVA, the lack of fit is insignificant, indicating that the second-order model with interaction is very adequate in approximating the response surface of the experimental design. The lack of fit's F-value of 3.4 implies that there is a 9.58% chance that a large lack of fit F-value could occur due to noise. A nonsignificant lack of fit is good. Applying multiple regression analysis, the results were fitted into a second-order polynomial  moisture, 0.3% (w/w) beef extract, and 0.05% (w/w) NaH2PO4. The final equation in terms of coded factors for the CCD model is expressed in  moisture, 0.2% (w/w) beef extract, and 0.0568% sodium dihydrogen phosphate. To verify the predicted result, a validation experiment was performed in triplicates. Under optimized conditions, the observed average experimental fibrinolytic enzyme production was 3750 ± 118 units/mL, indicating that the experimental and predicted values (3670 ± 48 units/mL) were in good agreement. The fibrinolytic activity obtained from the experiments was very close to the actual response predicted by the model, which proved the validity of the model.Based on the RSM result, the quadratic model predicted that the maximum production of fibrinolytic enzyme was 3705 units/mL, while the  Bacillus cereus IND1 fibrinolytic enzyme had a molecular mass comparable with that of the serine protease of Bacillus amyloliquefaciens DC-4 (28 kDa) [ Bacillus sp. DJ-4 (29 kDa) [ B. subtilis LD-8547 (30 kDa) [The enzyme was purified through four steps including ammonium sulfate precipitation, DEAE cellulose, gel filtration, and affinity chromatography. In the present study, the 30%–70% of saturated ammonium sulfate was found to be optimum for fibrinolytic enzyme precipitation. The recovery and purification were 4.57% and 12.8-fold, respectively, after casein-agarose affinity chromatography. The specific activity of the purified fibrinolytic enzyme was 960 units/mg protein. The purification summary is listed in (28 kDa) , Bacillu(29 kDa) , and B. (30 kDa) . Bacillus sp. KA38 metalloprotease [ B. subtilis strain A1 nattokinase [ B. subtilis natto B-12 [The results of optimum pH on enzyme activity and pH stability are shown in protease  and B. stokinase . The enz B. subtilis LD-8547 fibrinolytic enzymes [The plasminogen-rich plate showed 1.2-fold activity compared to plasminogen-free plate . These r enzymes . The deg B. cereus IND1. This organism effectively utilized wheat bran for the fibrinolytic enzyme production, and the process parameters were optimized by two-level full-factorial design and the RSM. This statistical approach proved to be a powerful tool for the optimization of fibrinolytic enzyme production. The optimal medium showed fourfold enzyme production compared to the unoptimized medium. Among the nutrient sources, sodium dihydrogen phosphate significantly increased enzyme production. The purified enzyme was active at higher temperatures and at wide pH ranges. It digested human blood clot in vitro. This study explores new sources of fibrinolytic enzymes to treat and prevent CVDs.A potent fibrinolytic enzyme was produced using agroresidues by


Macrobrachium nipponense, as the model animal for the semiconductor applied metals  to evaluate if the species is an suitable experimental animal of pollution in aquatic ecosystem.Acute toxicity testing were carried out the freshwater swamp shrimp, 50) of gallium, antimony, indium, cadmium, and copper for M. nipponense were estimated as 2.7742, 1.9626, 6.8938, 0.0539, and 0.0313 mg/L, respectively.The static renewal test method of acute lethal concentrations determination was used, and water temperature was maintained at 24.0 ± 0.5°C. Data of individual metal obtained from acute toxicity tests were determined using probit analysis method. The median lethal concentration (96-h LCM. nipponense with other species which exposed to these metals, it is obviously that the M. nipponense is more sensitive than that of various other aquatic animals.Comparing the toxicity tolerance of  Semiconductor manufacturing has become a leading industry in some developing countries. Gallium, antimony, indium, cadmium, and copper are essential transition metals that are widely used for the manufacture of integrated circuits, electroplating, and photoelectric appliances. These metals are released into the environment during manufacturing processes such as etching, wet polishing, and cleaning operations, which may produce much potentially hazardous waste, 2. AcciFreshwater animals can be used for controlling pollution in three ways, involving three different time frames: (1) the determination of water quality criteria from which standards can be established, (2) monitoring the health of populations of fish in the field or in a hatchery, (3) providing an early warning system for potential harm to the aquatic environment, 9. BothMacrobrachium nipponense) is a common aquatic invertebrate widely distributed in the downstream of rivers throughout the eastern Asia-Pacific[M. nipponense under laboratory conditions in order to determine specific safety concentrations.Freshwater swamp shrimp  of the five metals to M. nipponense, as presented in Table 50 values, the ranking of the five metals from most toxic to least toxic was: copper, cadmium, antimony, gallium, and indium.No mortality was detected in the control groups during laboratory static renewal tests (96-h acute toxicity) of the five metals. We recorded mortality in whole exposure durations for freshwater swamp shrimp, M. nipponense exposed to 0.5 mg/L within 96 hours  mg/L. No mortality was recorded in M. nipponense exposed to indium in 4.0 mg/L solution in the first 24 hours  mg/L for this metal. After 24 h, mortality was not observed for two of the metals (copper and cadmium) at concentrations of 0.05 mg/L  to be 14.32 mg/L; this reveals that M. nipponense are more sensitive to gallium exposure than tilapia larvae. Betoulle[Cyprinus carpio). Gallium also acts as a hepatotoxin and causes renal damage in treated C. carpio[Lin and Hwang estimate. carpio, 16.50 value of antimony for tilapia larvae (Oreochromis mossambicus) as 18.9 mg/L, and observed retardation in body growth at sublethal concentrations. The present study found M. nipponense to be more susceptible than tilapia larvae to acute antimony toxicity. With the exception of deleterious information on the effect on Daphnia magna, little or no research is available documenting the effect of antimony on freshwater invertebrates[Lin and Hwang reported50 value of indium for tilapia larvae (Oreochromis mossambicus) as 19.5 mg/L, demonstrating that tilapia larvae are more able to tolerate to indium exposure than M. nipponense. The indium value of 6.8938 mg/L was higher than that of gallium and antimony for the same stage of M. nipponense. This study is the first to examine the effects of indium on aquatic invertebrates.Lin and Hwang reported50 value of cadmium for white shrimp (Litopenaeus vannamei) is 1.07 mg/L; Tan and Wang[C. carpio exposed to sublethal concentrations of copper . Acute lethal effects of copper were attributed to excess mucous covering the gill tissues, leading to the breakdown of respiratory function[Both copper and cadmium are highly toxic for aquatic organisms; effects of short- and long-term exposure to these two metals have already been reported in a number of studies–22. Wu aand Wang also proand Wang reportedfunction.M. nipponense are frequently polluted by industrial wastewater. To maintain the balance of the local river ecosystems it is essential to know the susceptibility to these pollutants. We conclude that M. nipponense seem to be a promising candidate for evaluating freshwater quality, as they are sensitive to these metals. Moreover, no acute lethal effects were seen at concentrations of 0.28 mg/L , 0.20 mg/L (antimony), 0.69 mg/L (indium), 0.005 mg/L (cadmium), and 0.003 mg/L (copper). These findings are in good agreement with the concept of a safe level  as described by Sprague[M. nipponense of the same size.Regulation of different activities involving heavy metals does exist in an industrial country like Taiwan. HoweverMacrobrachium nipponense) were obtained from the local commercial suppliers. Shrimp were transported to a glass aquarium in our laboratory which was equipped with a water-cycling device; dechlorinated tap water  of 7.0 ~ 7.7 mg/L, and hardness of 38 ~ 45 mg CaCO3 /L) was used during the entire experiment. The temperature was maintained at 24.0 ± 0.5°C, with a 12-h light and 12-h dark photoperiod. Shrimp were acclimated for two weeks and fed an aquarium shrimp mixture every day. Shrimp (0.42 ± 0.17 cm in fork length) were used for acute toxicity tests in the initial experiments. Gallium sulfate (III) and antimony chloride (III) were purchased from Alfa Aesar . Indium chloride (III), copper sulfate (II), and cadmium chloride (II) were purchased from Sigma . All metal compounds had a purity of 99% or greater. Stock solutions were prepared in deionized water .Freshwater swamp shrimp (50) for M. nipponense. Ten animals of similar size were randomly sampled and placed in 10-L glass beakers. After 24 h of acclimatization, the M. nipponense were exposed to different concentrations of gallium , antimony , indium , cadmium , and copper  for 96 h or more. The control and each treated group were run in duplicate. During the experiment, dead animals were removed, with mortality recorded after 24, 48, and 96 h. The LC50 of every test chemical with 95% confidence limits were calculated for M. nipponense using a Basic program from the probit analysis described by Finney[Laboratory static renewal tests were conducted to determine the median lethal concentration (LCy Finney.


The study aimed to assess the possible systemic effects of intratympanic dexamethasone (IT-Dex) on the hypothalamic–pituitary–adrenal (HPA) axis, inflammation, and bone metabolism.A prospective cohort study including 30 adult patients of a tertiary referral ENT clinic treated with 9.6 mg IT-Dex over a period of 10 days was carried out.Effects on plasma and salivary cortisol concentrations  ACTH stimulation), peripheral white blood cell count, and biomarkers for bone turnover were measured before (day 0) and after IT-Dex (day 16). Additional measurements for bone turnover were performed 5 months after therapy. Clinical information and medication with possible dexamethasone interaction were recorded.P=0.58 and 0.24 respectively). Concentrations of osteocalcin (OC) and bone-specific alkaline phosphatase (BSAP) did not differ after dexamethasone ; similarly, there was no difference in the peripheral white blood cell count (5.7×1012/l and 6.1×1012/l on days 0 and 16 respectively).IT-Dex was well tolerated, and no effect was detected on the HPA axis (stimulated plasma and salivary cortisol concentration on day 0: 758±184 and 44.5±22.0 nmol/l; day 16: 718±154 and 39.8±12.4 nmol/l; IT-Dex therapy did not interfere with endogenous cortisol secretion or bone metabolism. Intratympanic glucocorticoids are effective in the treatment of sudden sensorineural hearing loss (SSNHL) In the treatment of SSNHL, a broad range of therapeutic modalities are being explored with different doses and applications of glucocorticoids that usually by far exceed the physiological daily cortisol production We therefore aimed to assess the possible systemic effects of IT-Dex injections on the HPA axis and bone metabolism, particularly on osteoblast function, as highly sensitive cells to exogenous glucocorticoids, in a prospective cohort study of patients receiving IT-Dex treatment.In our tertiary care hospital, a prospective study was carried out including adult patients with SSNHL or severe MD. All patients received IT-Dex treatment. Exclusion criteria were the following: inability to provide written consent, pregnancy or lactation, patients younger than 18 years, a history of systemic glucocorticoid treatment during the last 6 months, and a history of pituitary or adrenal disorders and liver disease.Local ethics committee approved the study protocol and an informed consent was obtained from each participant.IT-Dex was performed as described previously Measurements of basal plasma and salivary cortisol concentrations were performed immediately before IT-DEX (day 0), and 6 days after the last IT-Dex application, i.e. day 16 or 18 (referred to as day 16 in Low-dose (1 μg) ACTH (1-24)-corticotrophin was injected in order to generate a physiological adrenocortical stimulation Plasma and salivary cortisol levels were measured using a commercially available chemiluminescent immunoassay Cobas 6000 analyzer (Roche) according to the manufacturer's procedure. The interassay coefficient of variation was 4.5%. Thirty minutes after the injection of 1 μg ACTH, blood and saliva samples were taken to measure stimulated plasma and salivary cortisol concentrations respectively. The low-dose (1 μg) ACTH stimulation tests were carried out between 0800 and 0900. Samples were collected and analyzed immediately. Salivary samples were collected in Salivette The following additional blood samples of each patient were obtained on days 0 and 16 respectively: white blood cell count, osteocalcin (OC), and bone-specific alkaline phosphatase (BSAP). In order to evaluate any persistence of changes in BSAP over time, another blood test was carried out during outpatient clinical control 5 months after completing IT-Dex. Temperature-labile blood samples for OC were immediately sent for centrifugation and frozen until analyses were performed. The normal range for OC was considered to be between 8.8 and 29.7 μg/l for men and 5.2 and 34.5 μg/l for women. The normal range for BSAP in males was 6–30 μg/l, in premenopausal women 3–19 μg/l, and in postmenopausal women 6–26 μg/l. The plasma concentrations of OC and BSAP were measured by electrochemiluminescence immunoassays using a LIAISON Analyzer  for BSAP and by chemiluminescence immunoassay  for OC. The interassay coefficient of variation was 8.7% for BSAP and 6.05% for OC.Patients were interviewed on the day of the first IT-Dex application (day 0) and 6 days (day 16) after completion of the therapy. A detailed questionnaire relating to possible systemic side effects was completed by each participant after IT-Dex including mood changes, infections, particularly middle ear infections, trauma or surgery, and possible drug interactions with dexamethasone , as well as blood glucose control and blood pressure.www.graphpad.com). Data are expressed as means and s.d. unless stated otherwise. The paired T-test was used to assess normally distributed data (checked by the Kolmogorov–Smirnov test). The non-parametric Mann–Whitney test was applied otherwise. Relations between variables were analyzed by linear regression and Spearman's correlation analysis. A P value of <.05 was considered statistically significant in the above tests.Statistical analyses were performed using GraphPad Prism version 6.00 for Mac OS X, GraphPad Software, La Jolla, CA, USA  consecutive patients admitted to our ENT clinic with a mean age of 60.1±9.8 years. Baseline characteristics are given in P<0.001), i.e. before IT-Dex, and 458±214 and 718±154 nmol/l on day 16 (P<0.001), i.e. after IT-Dex . Similarly, there was no significant difference in basal and stimulated salivary cortisol levels before and after IT-Dex: 17.4±22.3 and 44.5±22.0 nmol/l on day 0, and 14.6±9.3 and 39.8±12.4 nmol/l on day 16 respectively . Basal and stimulated plasma cortisol concentrations were 461±236 and 758±184 nmol/l on day 0 . Similarly, levels of BSAP were 11.51±4.16 and 10.29±3.45 μg/l on days 0 and 16 respectively (P=0.278). Five months later, levels of BSAP (n=20) were 12.59±5.059 μg/l. No significant differences in BSAP were observed between day 0 and month 5 (P=0.558) and between day 16 and month 5 (P=0.134). Well-known age- and gender-related differences in OC and BSAP concentrations were observed in our cohort. The bone biomarker data are summarized in OC concentrations on day 0 were 24.1±10.5 μg/l and 23.6±8.8 μg/l on day 16, with no statistically significant difference before and after IT-Dex .The WBC remained within normal values on days 0 and 16, i.e. 5.7×10This study demonstrated that IT-Dex therapy in patients with SSNHL or severe MD does not adversely affect adrenal function or markers of bone metabolism. Intratympanic glucocorticoid application proved to be an effective treatment of SSNHL mainly because of higher endo- and perilymphatic glucocorticoid concentration compared with systemic glucocorticoid administration In conclusion, our study demonstrates that IT-Dex therapy does not cause systemic side effects, i.e. our findings confirm the normal function of the HPA axis and the osteoblasts after intratympanic application.


A reservoir with ecological and economic importance and its major tributary, localized in east-central Mexico, were studied. The aim of this work was to know the physicochemical water characteristics of both water bodies and to contrast these by their different uses, and also estimate overall water quality using a Water Quality Index (WQI). Water samples from the reservoir and the tributary were obtained in different climatic seasons. In the tributary, anoxic and hypoxic conditions and high levels of organic matter, orthophosphate, and ammonium showed that this is strongly impacted by wastewater discharges and that the water is not suitable for different uses; independently of the season, the WQI showed “poor” quality (34.4–47.2). In contrast, in the reservoir a better water quality was determined; the WQI in the sampling months ranged from 72.1–76.6 , and spatially, this was from 66.5–79.5 . In Mexico, there are approximately 4,462 man-made reservoirs and water retention berms, and it has been estimated that 150 billion cubic meters of water are stored in these reservoirs ,4,5,6,7,It is known that deterioration of reservoirs due to anthropogenic factors or natural processes affects the water quality, impairing its future use and also the protection of aquatic life ,11; in aCyprinus carpio comunis, Cyprinus carpio specularis, Cyprinus carpio rubrofuscus, Hypophtalmichthyus molitrix, Carassius auratus, and Ctenopharyngodon idellus) [C. carpio rubrofuscus and C. carpio specularis, for distribution throughout the state. The reservoir is also the habitat of a variety of aquatic wildlife [Ambystoma tigrinum; additionally, it represents an important site for shelter and nesting of resident birds, and many migratory ones find resources to live here [Furthermore, the Atlanga reservoir is utilized for the fishing of mainly the following six species of carp (idellus) , and it wildlife ,20, inclive here . In 2009ive here . Additioive here ,23.On the other hand, and owing to the difficulty in establishing water quality from many parameters, diverse authors have suggested the use of water quality indices (an efficient and simple monitoring tool), because a single number obtained from all of these parameters can indicate the water quality level ,11,24; aThus, the objective of the present work was to determine the physicochemical water characteristics of the Atlanga Reservoir and its major tributary (Zahuapan River), and to contrast these with the levels established for the purposes for which the reservoir is intended, such as agricultural irrigation, livestock, aquaculture, and protection of aquatic life; also estimate the overall quality of these water bodies by means of a Water Quality Index (WQI). It is expected that information from this work may be useful for decision making regarding the prevention and reduction of tributary pollution, prior to the reservoir reaching lower water quality levels.2)(w)w''(i')g, which is defined as mild with cool summer and long, sub-humid. The total annual precipitation is of 745.8 mm, December is the driest month and August the wettest, with 7.3 and 135.2 mm, respectively; January presents the highest number of frosts. The winds blow from North to South during the majority of the year [The Atlanga Reservoir is located at an altitude of 2,480 m above sea level and between latitudes 19°32' and 19°34' N and longitudes 98°12' and 98°09' W  in the Athe year ,26.The Atlanga Reservoir was inaugurated in 1961; it has a barrier of 24.2 m and includes a superficial area of 1,200 hectares, with a storage capacity of 54.5 million cubic meters and useful storage ranging from 40.9–48.5 million cubic meters . The maiT), transparency, and pH were determined in situ.At a site on the Zahuapan River , localizT, transparency, and pH were analyzed in situ. In the reservoir and during the same year, six samplings, corresponding to January, March, May, July, September, and November, were conducted. The samples were collected from the dam at eight sampling stations denominated as follows: Barrier (S1); Center (S2); Agricultural (S3); Airport Jetty (S4); Zahuapan Discharge (S5); San Luis (S6); Santa Clara (S7), and Fuerte Apache (S8), as depicted in 2Cr2O7) as oxidant (method 5220 D); DO by iodometric method (4500-O B); nitrite nitrogen (NO2−-N) by Griess reaction (method 4500-NO2-B), and phosphorus of orthophosphate (PO43−-P), by ascorbic acid methods (4500-P E) as reported by APHA-AWWA-WEF [3−-N) was analyzed by the Hach method (cadmium reduction), ammonium nitrogen (NH4+-N) with a selective electrode , and hardness ) as indicated in Mexican Norm [T, and for pH, a potentiometer  previously calibrated with 4.0 and 7.0 buffers; while reservoir depth was determined with a portable sounder .Samples were analyzed for several parameters. Total suspended solids (TSS) were calculated for weight differences after drying at 105 °C (method 2540 D); organic matter (OM), such as chemical oxygen demand, utilized the closed reflux method with potassium dichromate , the WQI reported by Pesce and Wunderlin [For the individual parameters, means and standard deviation (SD) were obtained. The means were compared by one-way Analysis of variance (ANOVA); when significant underlin , and widunderlin ,32, was The parameters normalization and assigned weights for the WQI are according to values proposed for aquatic life preservation ; the watCi) by assigning values ranging from 0–100% as was previously reported [Ci 100 was assigned from twice 50 cm, the upper value established for carp, and of 70 to 50 Ci values, corresponding to 50–30 cm, the range established for this fish. Additionally, each parameter was assigned a relative weight (Pi) within a range from 1–4, where 1 is assigned to the parameter least important for aquatic life preservation, and 4, the most important; in this case, number 4 was assigned to DO, number 3 to NH4+-N and to OM, number 2 to NO2−, NO3− and transparency, and number 1 to hardness, pH, PO43−, and to T. Finally, for the k constant, which represents the visual appearance concerning the contamination of the water body and that could be evaluated by an individual without training in environmental knowledge, the value assigned was 1; when k receives this value, the index is known as the objective WQI [To calculate the WQI, the concentration of each parameter (except transparency) was normalized  . In March, July, and September, this parameter was 14.9 ± 1.9, 14.9 ± 1.1, and 14.9 ± 0.4 °C, respectively, despite the seasonal difference. pH had a similar pattern with a increase in May, with a value of 8.3 ± 0.40 , and in March, July, and September, this remained nearly neutral . Anoxic conditions in March and May were determined; in July, OD was 4.4 ± 0.26 mg/L and no significant differences were observed across the sampling schedules. In September, hypoxic conditions were determined in the first 6 h, with an average of 0.9 ± 0.75 mg/L. Differences were found between these months , although both belonged to the rainy period.The results are presented in 2/L); even when DO was present in July, this registered similar concentrations to those of the previous months . In September, the OM concentration decreased (122.2 ± 44.0 mgO2/L), probably due to an increase in river flow. Otherwise, in May, TSS concentration was 802 ± 726 mg/L, and in July, this concentration was duplicated . In both months, the high content may be caused by the beginning of the rainy season and the heavy rains, which drag the accumulated solids into the riverbed and onto the surrounding land. The lowest level of solids was established in September with 29.9 ± 22.8 mg/L, and means found in the months were statistically different . Additionally, inorganic solids predominated (≈80%) at all times.The OM concentration presented high variation between the schedules and exhibited the highest levels from March to July (means > 200 mgOT and pH fell within the permissible limits according to Protection of Aquatic Life (PAL) and Agricultural Irrigation (AI), as established by the Mexican Norm [can Norm . Howevercan Norm , coincidcan Norm . It is kcan Norm . DO levecan Norm  and withcan Norm . OM was can Norm . Also, tcan Norm , or watecan Norm . 4+-N was the predominant form and the means were significantly different ; in March, we found 20.2 ± 1.7 mg/L, while in May, this level increased in the scheduled samplings to 49.5 mg/L, with a mean of 35.9 ± 7.9 mg/L; Tukey (p < 0.05) showed differences. In July and September, and apparently due to the rainy season (p > 0.05). The existence of NH4+ in water is an indicator of direct pollution [4+-N/L recommended [et al. [With regard to the inorganic nitrogen analyzed, NHy season b, the coollution , and it ommended , surpass [et al. .2−-N, were not present during the first two months, while in July and September, these were 0.02 and 0.056 mg/L, respectively, and differences were found . NO3−-N was not detected in March; in May and September, it was registered at low levels (<0.1 mg/L) due to the lack of or low DO concentration, which did not favor NH4+-N oxidation, while in July this reached 0.22 mg/L. NO2−-N and NO3−-N levels were lower than 10 and 90 mg/L, maximum limits established for livestock use, respectively [Oxidized forms, such as NOectively .43−-P concentrations were highest in March (3.7 ± 0.27 mg/L), followed by May with 2.6 ± 0.46 mg/L , and these decreased in the following months to minimum values (0.62 ± 0.18 and 0.78 ± 0.05 mg/L), during which the concentrations were not statistically different . All of the concentrations determined were >0.1 mg/L of phosphates, the maximal level permitted to control the accelerated eutrophication, in rivers and streams [43−-P may be largely associated with wastewater discharges containing detergents [Phosphorus is a nutrient that can promote the development of species that accelerates the eutrophication process. In this tributary, PO streams ; the higtergents , althougF = 21.174; p < 0.05; Tukey, p > 0.05), and decreased in July (225.5 ± 15.2 mg/L), exhibiting significant differences with respect to the remaining months . Among sampling stations, Stations S1 and S2 had the greatest depth (around 6.0 m), and Stations S4 to S6 exhibited shallow conditions, although the S5 sampling station (Zahuapan Discharge) presented the lowest level (0.9 ± 0.3 m); this could be due to hauling solids through the river and subsequent release into the reservoir, for example, 1,529 ± 536 mgTSS/L  and was higher from May to September (21.0 ± 0.4 to 23.4 ± 0.7 °C), corresponding to the warmer season, and was lower in the remaining months (16.8 ± 1.2 to 17.6 ± 0.7 °C) according to the seasonal profile, as exhibited at other reservoirs [T ranged from 19.1 ± 3.2 to 20.6 ± 2.3 °C. The levels found for this parameter are suitable for the different water uses [Water ter uses .F = 19.064; p < 0.05); January and May demonstrated alkaline conditions with levels ≥8.3 , followed by March and July, which ranged from 7.3 ± 0.1 to 7.9 ± 0.04. In November, a wide variation was determined due to the alkaline tendency at six sampling stations and to the acidic conditions found at stations S4 and S5, which reached minimal values of 6.0. Chapman and Kimstach [Throughout all of the months, the pH presented significant variation . In March, July, and November, the reservoir exhibited highest levels (7.4 ± 0.4 to 7.6 ± 0.7 mg/L) and these were similar , while in January, concentration diminished and there was high variation among stations (6.4 ± 0.9 mg/L). In May and September, levels were lower , and at this time, stations S4 and S5 registered levels <5.0 mg/L, not suitable for PAL and carp aquaculture [Regarding DO concentration, this presented monthly significant differences , and January and March achieved acceptable concentrations . In May, these levels diminished to 4.4 ± 6.2 mgO2/L and exhibited good quality, while in July and November, the levels increased to 41.7 ± 18.1 and 80.7 ± 13.5 mgO2/L, respectively, and showed a significant difference , reaching levels of polluted water [2/L. A significant diminution with respect to that found in the tributary was observed at Station S5, probably due to the partial degradation of organic matter; consequently, a negative effect on DO and pH at this station was observed.Significant differences were found for OM (p < 0.05); in July, an increase was detected (206.1 ± 9.2 mg/L). During these months, the levels of this parameter were found among those of polluted water [TSS content showed similar concentrations in March and May 160 mg/L) ; in July and September, this decreased to 12.8 ± 3.4 and 14.8 ± 1.8 cm, respectively. Sampling stations exhibited levels from 15.6 ± 4.1 to 18.2 ± 3.7 cm, demonstrating high variation. Transparency levels established for aquaculture  range from 30–50 cm [Transparency was similar from January to May , while in January and May, we found concentrations of 0.12 ± 0.07 and 0.07 ± 0.06 mg/L, respectively, exceeding the limit permitted for use in PAL [In terms of NHe in PAL . From Ju2−-N was present at low and similar concentrations , with a significant increase between March and July (0.023 ± 0.004 to 0.037 ± 0.02 mg/L); at sampling stations, concentrations ranged from 0.011 ± 0.015 to 0.023 ± 0.024 mg/L. From January to May, NO3−-N was not found; in July and November, it was detected (0.48 ± 0.6 and 0.02 ± 0.03 mg/L) and there was not a significant difference , but a significant increase was found in September (2.7 ± 1.7 mg/L). At the sampling stations, the concentrations of this parameter ranged from 0.14 ± 0.2 to 0.88 ± 1.8 mg/L, and Stations S1 and S2 exhibited the lowest concentrations. To both parameters, all of the levels were optimal for livestock [In January, September, and November, NOivestock .43−-P concentration exhibited a significant difference among the months , with January presenting the lowest concentration (0.17 ± 0.01 mg/L), followed by March and May, which had similar levels . An increase was observed in July, reaching 0.48 ± 0.05 mg/L. In November, the level decreased to 0.32 ± 0.02 mg/L. Among sampling stations, homogeneous concentrations was presented and ranged from 0.25 ± 0.10 to 0.31 ± 0.15 mg/L. PO43−-P levels in the sampled months and at the stations were >0.025 mgP/L, the maximal limit established for dams [The POfor dams . This reF = 159.42; p < 0.05); 126.2 ± 5.3 mg/L were found in January, and in March and May, the concentrations showed no significant difference . From July to November, highest concentrations were reached , and there were differences among these . Sampling stations presented concentrations ranging from 197.0 ± 66.4 to 205.0 ± 66.4 mg/L. The water was classified as hard and very hard [Water hardness was different among the sampling months  to 79.5 , as depicted in Lowest WQI value was found for Station S5, the site of the discharge of the tributary (72.9 ± 3.2) and that exhibited typical characteristics of the riverine zone in the reservoirs, because this zone receives a high input of nutrients ; this coIn the reservoir during the months studied, “good” water quality was determined (72.1–76.6) b, and un4+-N or PO43−-P, the water quality did not improve significantly.In the tributary, the WQI reached values from 34.4–38.9, corresponding to March, May, and September; in July, a slight increase was observed (47.2), probably due to the increase in DO and the increase of rainfall, which acted as a dilution factor, although all calculated levels fell within the range of “poor” water quality b. These Scirpus lacustris L.), could be an especially strong contributor because there is a strip on the edge of the reservoir and observations in the study area have revealed a higher abundance of this plant in recent years. Regardless of this, this plant appears to entertain a duality, because if it continues growing so excessively, it can have adverse effects on the site in terms of gaining ground and contributing to eutrophication. Notwithstanding this, the following have been observed: (1) the tule has served to shelter birds and their nests, and it is the habitat of other aquatic organisms [4+ levels are high and additional oxygen is necessary for their oxidation into CO2 and NO3─, respectively [However, when the water from the tributary was released into the reservoir, an important increase in the WQI was observed; the “poor” quality of the tributary was reverted in the reservoir, probably due to the following different factors: 1) the contribution of primary producers (by nutrient utilization and oxygen production), among these phytoplankton, although aquatic vegetation, such as the tule ectively ; as wellectively ; conversFinally, in order to know the evolution of water quality in the reservoir and its tributary, from parameters reported by two studies ,43, in s43−-P, and NH4+-N showed that the tributary was strongly impacted by wastewater discharge; due to the levels found in several parameters, the water is not suitable for PAL and in some time for AI uses, and the WQI reported “poor” quality (34.4–47.2), independent of the season. In the reservoir, the physicochemical conditions showed better levels, due to the contribution of biological and physical factors, and the WQI during the sampling months ranged from 72.1–76.6 , while spatially, this ranged from 66.5–79.5 , and 10% of these values were found at <70%. Due to all of the latter, and although the reservoir presented best water quality, it is necessary to pay attention to the tributary (in order to reduce and avoid pollution), before the reservoir reaches lower water quality levels and exerts a negative impact on the activities of protection of the aquatic life, irrigation, fisheries, and aquaculture, practiced at the reservoir. In the tributary, anoxic and hypoxic conditions and high levels of OM, PO