Marker-free genomic editing in Saccharomyces cerevisiae using universal donor templates and multiplexing CRISPR-CAS9.

The budding yeast Saccharomyces cerevisiae is an excellent model organism for studying a variety of critical cellular processes. Traditional methods to knock in or -out at specific yeast loci utilize polymerase chain reaction-based techniques, in which marker cassettes with gene-specific homologies are integrated into the genome via homologous recombination. While simple and cost-effective, these methods are limited by marker availability when multiple edits are desired. More recently, CRISPR-Cas9 technology has introduced methods to edit the yeast genome without the need for selectable markers. Although efficient, this method is hindered by additional reagents and lengthy protocols to design and test unique guide RNAs and donor templates for each desired edit. In this study, we have combined these two approaches and have developed a highly efficient economical method to edit the yeast genome marker-free. We have designed two universal donor templates that efficiently repair commonly used selectable markers when targeted by a novel guideRNA-Cas9 designed to promoter regions in Ashbya gossypii found in most integration modules. Furthermore, we find our newly designed guideRNA-Cas9 successfully multiplexes when multiple markers are present. Using these new tools, we have significantly improved the cost and efficiency to generate single or multiple marker-free genetic modifications. In this study, we demonstrate the effectiveness of these new tools by marker-free ablating PRC1, PEP4, and PRB1 vacuolar proteases typically inactivated before many biochemical and membrane-trafficking studies using budding yeast.

Prediction of Alzheimer's Disease Based on 3D Genome Selected circRNA.

Alzheimer's disease (AD) is a neurodegenerative disease and there is by far no effective treatment for it, especially in its late stage. Circular RNAs (circRNAs), known as a class of non-coding RNAs are widely observed in eukaryotic transcriptomes, and are reported to play an important role in neurodegenerative diseases including AD. circRNAs usually act as microRNA (miRNA) inhibitors or «sponges¼ to regulate the function of miRNAs, leading to subsequent changes in protein activities and functions. Accumulating evidence indicates that circRNAs can serve as potential biomarker in AD early prediction. The functional roles of circRNAs are very versatile including miRNAs binding - thus affecting downstream gene expression, generating abnormally translated protein peptides, and affecting epigenetic modifications which subsequently affect AD related gene expressions. Therefore, identifying AD-related circRNAs can contribute to AD early diagnosis and intervention. In this work, we collected and curated an AD-related circRNA dataset; by exploring the circRNAs' corresponding DNA loci distribution in chromatin 3D conformation (3D genome) and utilize the such 3D genome information, we were able to selected a concise yet predictively effective circRNA panel, based on which, significantly better AD prediction machine learning models were achieved.

[Development and validation of three-dimensional body image stimuli for Chinese middle-aged adults].

Objective: To develop a three-dimensional body image stimuli suitable for middle-aged people in China, and verify the validity and reliability of the body image stimuli. Method: According to China and World Health Organization body mass index classification standards of adults, a set of three-dimensional body image stimuli of Chinese middle-aged males and females with different body size was developed by using 3D Studio Max and Adobe Photoshop CC based on the literature and expert consultation method. Forty-two 45- and 59-year-old middle-aged people in Zhengzhou City, Henan Province were recruited to verify the three-dimensional body image stimuli. Through questionnaire survey and physical examination, the coincidence between the selected body type and the actual body type was tested; the body composition was measured by dual-energy absorptiometry (DXA), and the structure validity of the image was tested; the body size satisfaction was investigated by the body image stimuli and the standard questionnaire, and the empirical validity of the image was tested. The repeated survey was conducted 14 days after the initial survey, and three experts were invited to score the current somatotype of the subjects to test the test-retest reliability and inter-rater reliability of the body image stimuli. Pearson, Spearman, Kendall correlation and Kappa consistency analysis were used to evaluate the validity and reliability of the body image stimuli. Results: The average age of 42 subjects was 52.7 years old, including 13 males and 29 females. A group of three-dimensional body image stimuli of middle-aged men and women were developed, and each group included 8 images. 73.8% of the subjects chose the body size consistent with the actual body type, and the weighted Kappa coefficient was 0.755 (P<0.01). The selected somatotype was positively correlated with body weight and body composition indexes such as fat content, and the Pearson correlation coefficient of construct validity was 0.623-0.717 (P<0.05). The results of the two surveys were positively correlated, and the Spearman correlation coefficient of test-retest reliability was 0.784-0.821 (P<0.05). The scores of the three experts on the current somatotype of the subjects were positively correlated, and the Kendall correlation coefficient of inter-rater reliability was 0.818-0.878 (P<0.05). Conclusion: The development of principle and reference basis of three-dimensional body image stimuli of middle-aged people is reliable, and the validity and reliability of the body image stimuli are good.

[Short-term effects of household indoor- and outdoor-originating fine particulate matters on heart rate and heart rate variability in chronic obstructive pulmonary diseases patients, Beijing].

Objective: To assess the associations of indoor fine particulate matter (PM(2.5)) from outdoor and indoor sources with heart rate (HR) and heart rate variability (HRV) in patients with chronic obstructive pulmonary disease (COPD) of Beijing. Methods: A total of 40 male patients in a stable stage of COPD were recruited from a hospital in a panel study in Beijing with 5 consecutive days of measurement for each subject. General information and disease history of the participants from questionnaires were obtained prior to the study. HR and HRV were repeatedly examined using dynamic electrocardiograph. HRV included standard deviation of all NN intervals (SDNN), root mean square of successive differences between adjacent NN intervals (rMSSD), total power (TP) power in the low-frequency band (LF) and the high-frequency band (HF). Iron was used as tracer element to separate indoor-originated PM(2.5) and outdoor-originated PM(2.5). Mixed-effect models were applied to assess the associations of outdoor-originated PM(2.5) or indoor-originated PM(2.5) and health effects. Results: The P(50) (P(25), P(75)) values of daily indoor PM(2.5), indoor-originated PM(2.5) and outdoor-originated PM(2.5) were 50.9 (26.8, 122.7), 16.0 (1.9, 43.7) and 27.3 (13.5, 61.8) µg/m(3), respectively. The mean±SD of concentrations of real-time indoor PM(2.5), indoor-originated PM(2.5) and outdoor-originated PM(2).5 were (61.5±58.8), (25.3±39.1) and (36.2±42.7) µg/m(3), respectively. Compared with outdoor-originated PM(2.5), indoor-originated PM(2.5) had significant associations with HRV and HR. Each 10 µg/m(3) increase at 4 h indoor-originated PM(2.5) and outdoor-originated PM(2.5) moving average was associated with 3.4% (95%CI: -4.7%, -2.1%) and 0.6% (95%CI: -2.0%, -0.8%) reduction in TP (P<0.001). Each 10 µg/m(3) increase at 12 h indoor-originated PM(2.5) moving average was associated with 7.6% (95%CI: -10.1%, -5.1%), 4.7% (95%CI: -6.7%, -2.7%), 3.3% (95%CI: -4.2%,-2.4%) and 3.0% (95%CI: -4.5%, -1.5%) reduction in HF, LF, SDNN and rMSSD, respectively. Each 10 µg/m(3) increase at 12 h outdoor-originated PM(2.5) moving average was associated with 0.7% (95%CI: -2.7%, -1.4%), 0.2% (95%CI: -1.9%, 1.4%), 0.7% (95%CI: -1.4%, -0.1%) and 0.2% (95%CI: -1.3%, 0.9%) reduction in HF, LF, SDNN and rMSSD, respectively (P<0.001). Each 10 µg/m(3) increase at 8 h indoor-originated PM(2.5) and outdoor-originated PM(2.5) moving average was associated with 0.7% (95%CI: 0.4%, 1.0%) and 0.4% (95%CI: 0.2%, 0.6%) increase in HR. Conclusion: Exposure to indoor-originated PM(2.5) was more strongly associations with HRV indices and HR compared with outdoor-originated PM(2.5) in male COPD patients.

[Effects of high-efficiency particulate air purifiers on indoor fine particulate matter and its constituents in a district of Beijing during winter].

OBJECTIVE: To analyze the effect of domestic high-efficiency particulate air (HEPA) purifiers on the concentrations of indoor fine particulate matter (PM2.5) and its elementary constituents in 20 residences in a district of Beijing during winter. METHODS: From November 2015 to January 2016, 20 residences in a district of Beijing were selected, where indoor and outdoor PM2.5 data were collected simultaneously in three time periods according to the operating of air purifiers (Group 0 h: 24 hours before operating; Group 24 h: 24 hours after operating; Group 48 h: 24 to 48 hours after operating). The content of 21 elements in PM2.5 samples were determined by inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma optical emission spectrometry (ICP-OES). Indoor/outdoor particle concentration ratio (I/O ratios) and ΔI/O ratios were used to describe the pollution levels and the variation range of PM2.5 and its 21 elementary constituents. One-way analysis of variance (ANOVA) for repeated measurement data was applied to compare the I/O ratios of PM2.5 and its elementary constituents among the different groups, and Bonferroni method was used for comparison in pairs. Wilcoxon signed rank test for paired-samples was used to compare ΔI/O ratios of 21 elementary constituents with that of PM2.5. RESULTS: The median I/O ratios of PM2.5 in the three groups were 1.27 (P25-P75: 0.50-2.68), 0.45 (P25-P75: 0.27-1.03) and 0.36 (P25-P75: 0.28-2.48), respectively. Compared with Group 0 h, the I/O ratios of PM2.5 in Group 24 h (P=0.042) and Group 48 h (P=0.006) decreased significantly. However, there was no significant difference between Group 24 h and Group 48 h. Significant differences were found comparing ΔI/O ratios of aluminium, ferrum and titanium to that of PM2.5, in both Group 24 h and Group 48 h (P<0.05). No significant change was found in the I/O ratios of these three elements among the three groups before and after air purifier operating (P>0.05). Distances from residences to traffic arteries could affect I/O ratios of some elements from traffic-related source (P<0.05). CONCLUSION: Domestic HEPA air purifiers could effectively reduce indoor PM2.5 concentration, and the pollution level of PM2.5 tend to be stable after the purifier operating for a time. The purifiers had different effects on different elements, among which most showed statistical significances.

Multidrug-resistant tuberculosis patients' views of interventions to reduce treatment loss to follow-up.

SETTING: Patients who initiated treatment for multidrug-resistant tuberculosis (MDR-TB) at 15 Programmatic Management of Drug-resistant Tuberculosis (PMDT) health facilities in the Philippines between July and December 2012. OBJECTIVES: To describe patients' views of current interventions, and suggest changes likely to reduce MDR-TB loss to follow-up. METHODS: In-depth interviews were conducted between April and July 2014 with MDR-TB patients who were undergoing treatment, had finished treatment at the time of the interview (controls), or had been lost to follow-up (LTFU). Responses were thematically analyzed. RESULTS: Interviews were conducted with 182 patients who were undergoing or had completed treatment and 91 LTFU patients. Views and suggestions could be thematically categorized as approaches to facilitate adherence or address barriers to adherence. The top themes were the need for transportation assistance or improvements to the current transportation assistance program, food assistance, and difficulties patients encountered related to their medications. These themes were addressed by respectively 63%, 60%, and 32% of the participants. CONCLUSIONS: A more patient-centered approach is needed to improve MDR-TB treatment adherence. Programs should strive to provide assistance that considers patient preferences, is adequate to cover actual costs or needs, and is delivered in a timely, uninterrupted manner.

Multidrug-resistant tuberculosis patients lost to follow-up: self-reported readiness to restart treatment.

SETTING: Multidrug-resistant tuberculosis (MDR-TB) patients lost to follow-up (LTFU) from Programmatic Management of Drug-resistant Tuberculosis facilities in the Philippines. OBJECTIVES: To gain insight into patients' readiness to return to treatment. METHODS: MDR-TB patients who initiated treatment and were categorized as LTFU were identified using TB registers, contacted, and asked to consent to an interview and medical record review. At the conclusion of the interview, patients' readiness to restart treatment was assessed and examined in relation to demographic, clinical, and interview data. Odds ratios were calculated. RESULTS: When asked if they would consider restarting MDR-TB treatment, 3% of the 89 participating patients reported that they had already restarted, 34% indicated that they wanted to restart, 33% had not considered restarting, 28% were undecided, and 2% had decided against restarting. Patients who wanted to restart treatment were more likely to report having borrowed money for TB-related expenses (OR 5.97, 95%CI 1.27-28.18), and were less likely to report being self-employed (OR 0.08, 95%CI 0.01-0.67), or perceive themselves at low or no risk for TB relapse (OR 0.30, 95%CI 0.08-0.96) than patients who did not indicate an interest in restarting treatment. CONCLUSIONS: Efforts to re-engage LTFU patients in care should consider financial barriers, knowledge gaps, and personal adherence challenges in patients.

Responding to the multidrug-resistant tuberculosis crisis: mainstreaming programmatic management to the Philippine National Tuberculosis Programme.

SETTING: The Philippines ranks eighth among 27 priority countries for multidrug-resistant TB (MDR-TB). OBJECTIVE: To describe a model of public-private partnership in MDR-TB management. METHODS: An exploratory study of integrating MDR-TB management initiated in private-public mix DOTS into the National TB Programme (NTP). RESULTS: Recognising that MDR-TB was a threat to DOTS, the Tropical Disease Foundation initiated MDR-TB management in 1999. An official mandate for the integration of MDR-TB services into the NTP was issued by the Department of Health in 2008. With an increased government budget augmented by support from the Global Fund to Fight AIDS, Tuberculosis and Malaria, 1294 MDR-TB patients were placed on treatment from 1999 to 2008. The treatment success rate improved from 64% in 1999 to 75% in 2005. There are now five MDR-TB treatment centres with 181 treatment sites in Metro Manila, and three culture centres. People trained include 12 master trainers, 31 trainers, 25 treatment centre and 381 treatment site staff. CONCLUSION: Mainstreaming into the NTP of this unique model of MDR-TB management through a dynamic public-private collaboration can be considered best practice in implementation science of an evidence-based intervention leading to change in health care policy and practice.

A prospective study of pulmonary function in Hodgkin's lymphoma patients.

BACKGROUND: To prospectively study changes in lung function in Hodgkin's lymphoma (HL) patients and to explore predictors for these changes over time. METHODS: In all, 52 patients with HL receiving bleomycin-based chemotherapy with (n = 23) or without (n = 29) mediastinal radiotherapy were enrolled. Pretreatment pulmonary function tests were carried out. These were repeated at 1 month, 6 months, and 1 year after therapy. RESULTS: With chemotherapy alone, the median %DLCO declined significantly at 1 month but returned to baseline by 6 months. The median %DLCO did not further decrease with radiotherapy, but remained persistently reduced at 1 year. In patients who received radiotherapy, having >33% of lung volume receive 20 Gy (V20) and a mean lung dose (MLD) of >13 Gy significantly predicted for persistently reduced %DLCO at 6 months (P = 0.035). Smoking significantly predicted for a persistently reduced %DLCO at 1 year (P = 0.036). On multivariable analysis, significant predictors for decline in %DLCO at 1 year were higher baseline %DLCO (P = 0.01), higher MLD (P = 0.02), and a smoking history (P = 0.02). CONCLUSIONS: Several factors contribute to decline in %DLCO in HL patients who received bleomycin-based computed tomography. The identification of threshold radiation dosimetric parameters for reduced lung function may provide guidance in the radiation planning of these patients.

Properties of Cu(thd)2 as a precursor to prepare Cu/SiO2 catalyst using the atomic layer epitaxy technique.

The new Cu/SiO2 catalyst is developed by the atomic layer epitaxy (ALE) method. The ALE-Cu/SiO2 catalyst with high dispersion and nanoscale Cu particles appears to have very different catalytic properties from those of the typical Cu-based catalysts, which have satisfactory thermal stability to resist the sintering of Cu particles at 773 K. Due to the formation of small Cu particles, the ALE-Cu/SiO2 can strongly bind CO and give high catalytic activity for CO2 converted to CO in the reverse water-gas-shift reaction. The catalytic activity decreases in the order of 2.4% ALE-Cu/SiO2 =... 2% Pt/SiO2 > 2% Pd/SiO2 > 10.3% IM-Cu/SiO2.

OPAAS: a web server for optimal, permuted, and other alternative alignments of protein structures.

The large number of experimentally determined protein 3D structures is a rich resource for studying protein function and evolution, and protein structure comparison (PSC) is a key method for such studies. When comparing two protein structures, almost all currently available PSC servers report a single and sequential (i.e. topological) alignment, whereas the existence of good alternative alignments, including those involving permutations (i.e. non-sequential or non-topological alignments), is well known. We have recently developed a novel PSC method that can detect alternative alignments of statistical significance (alignment similarity P-value <10(-5)), including structural permutations at all levels of complexity. OPAAS, the server of this PSC method freely accessible at our website (http://opaas.ibms.sinica.edu.tw), provides an easy-to-read hierarchical layout of output to display detailed information on all of the significant alternative alignments detected. Because these alternative alignments can offer a more complete picture on the structural, evolutionary and functional relationship between two proteins, OPAAS can be used in structural bioinformatics research to gain additional insight that is not readily provided by existing PSC servers.

Modulation of immune response following dietary genistein exposure in F0 and F1 generations of C57BL/6 mice: evidence of thymic regulation.

To further determine whether genistein (GEN) modulation of the immune responses was related to its endocrine-disrupting properties and time of exposure, pregnant C57BL/6 mice were exposed to GEN at 0-1250 ppm in feed starting on day 14 of gestation. The C57BL/6 offspring were exposed to GEN in utero and lactationally, and through feed after weaning until postnatal day 42. In dams, exposure to GEN increased the terminal body weight (250 and 1250 ppm), the number of splenic T cells and NK cells (250 ppm), and the activity of NK cells (250 ppm). In F(1) males, GEN increased the terminal body and spleen weights (25 and 250 ppm), the number of CD4(+)CD8(+) and CD4(-)CD8(+) thymocytes (25 ppm), and the number of splenic T cell subsets and NK cells (25 and 250 ppm). Moreover, splenic NK cell activity and anti-CD3-mediated splenocyte proliferation were increased in all treatment groups. In F(1) females, the percentages of CD4(-)CD8(+) and CD4(-)CD8(-) thymocytes (25 and 250 ppm), and CD4(+)CD8(-) and CD4(+)CD8(+) splenocytes (25 and 250 ppm) were increased. In contrast, the percentage and number of CD4(+)CD8(+) thymocytes were decreased (250 ppm). Exposure to GEN decreased the percentages of splenic NK cells in all treatment groups, and decreased the activity of splenic NK cells at the 25 ppm concentration. Additionally, evaluation of CD25(+) and CD44(+) expression by thymocytes indicated that the decrease in the percentage of CD44(+)CD25(+) thymocytes was at least partially responsible for the decrease in the percentage of CD4(-)CD8(-) thymocytes in F(1) male mice. Overall, the results demonstrate that GEN can modulate the immune system in both adult and developing C57BL/6 mice in a dose-specific manner. The gender-specific effects of GEN on the immune responses in F(1) mice suggest that GEN may modulate the immune system by functioning as either an estrogen agonist or antagonist. The differential effects of GEN on thymocytes in F(1) male and female mice indicate that GEN immunomodulation might be related to its effect on thymus.

Kinetics of inorganic carbon utilization by microalgal biofilm in a flat plate photoreactor.

A kinetic model was developed to describe inorganic carbon utilization by microalgae biofilm in a flat plate photoreactor. The model incorporates the fundamental mechanisms of diffusive mass transport and biological reaction of inorganic carbon by microalgal biofilm. An advanced numerical technique, the orthogonal collocation method and Gear's method, was employed to solve this kinetic model. The model solutions included the concentration profiles of inorganic carbon in the microalgal biofilm, the growths of suspended microalgae and microalgal biofilm, the effluent concentrations of inorganic carbon, and the flux of inorganic carbon from bulk liquid into biofilm. The batch kinetic test was independently conducted to determine biokinetic parameters used in the microalgal biofilm model simulation while initial thickness of microalgal biofilm were assumed. A laboratory-scale flat plate photoreactor with a high recycle flow rate was setup and conducted to verify the model. The volume of photoreactor is 60 l which yields a hydraulic retention time of 1.67 days. The model-generated inorganic carbon and the suspended microalgae concentration curves agreed well with those obtained in the laboratory-scale test. The fixation efficiencies of HCO(3)(-) and CO(2) are 98.5% and 90% at a steady-state condition, respectively. The concentration of suspended microalgal cell reached up to 12 mg/l at a maximum growth rate while the thickness of microalgal biofilm was estimated to be 104 microm at a steady-state condition. The approaches of experiments and model simulation presented in this study could be employed for the design of a flat plate photoreactor to treat CO(2) by microalgal biofilm in a fossil-fuel power plant.

The Autosow raised miniature swine as a model for assessing the effects of dietary soy trypsin inhibitor.

Toxicological effects of dietary soy trypsin inhibitor (TI) were assessed in male miniature swine, a model chosen for its similarities to human digestive physiology and anatomy. The TI preparation was extracted from defatted raw soy flour. From 1 through 5 weeks of age, piglets were automatically fed either a TI liquid diet [Autosow TI group (ASTI)] or a control liquid diet [Autosow control group (ASC)]. From 6 to 39 weeks of age, these animals received either swine chow and TI or swine chow and control article. The TI diets were formulated to contain a TI activity of approximately 500 mg TI/100 g dry matter. A sow control (SC) group suckled from birth to 6 weeks of age and then fed as the ASC group with swine chow plus control article from 6 to 39 weeks of age. The SC piglets grew faster than ASC piglets during postnatal weeks 1 and 2; however, the ASC piglets were significantly heavier than the SC piglets (P=0.001) at 6 weeks of age. Compared with the ASC group, TI caused a moderate decrease in feed consumption and a moderate but reversible decrease in growth from 2 to 5 weeks of age, but not thereafter. Some control and TI-fed Autosow-reared piglets had loose stools until 6 weeks of age; the effect was significantly greater in the TI-fed group. Otherwise, all swine were active and had normal appearance and behavior.

Pathological evaluation, clinical chemistry and plasma cholecystokinin in neonatal and young miniature swine fed soy trypsin inhibitor from 1 to 39 weeks of age.

The potential toxicity of dietary soy trypsin inhibitor (TI) was evaluated in neonatal miniature swine. From 1 to 6 weeks of age, two groups of male piglets were artificially reared in an Autosow and automatically fed either TI or control liquid diet. From 6 to 39 weeks of age, these two groups were fed either TI or control chow diet. A third group, sow control (SC), suckled from birth to 6 weeks of age, were also weaned to control chow from 6 to 39 weeks of age. Clinical chemistry and plasma cholecystokinin (CCK) determined at 6, 18, 30 and 39 weeks of age, and serum amylase activity with gross and histopathological analyses of major organs at 6 and 39 weeks of age are reported. TI had no effect on plasma CCK, serum amylase activity, or numerous clinical chemistry values. TI-fed piglets had a larger relative liver weight at 6 weeks of age. Relative pancreas weight decreased with age but was not affected by TI. Gross and histopathological analyses of major organs, except the spleen, were within normal limits. Increased incidence of extramedullary hematopoiesis was noted in the spleen of the TI group at 6 but not at 39 weeks of age. There was no consistent pattern in immunohistochemical foci for secretin, gastrin releasing polypeptide or CCK, and no change in DNA, RNA, mitotic index or nuclear density of pancreatic cells. At 6 weeks of age, TI increased pancreatic protein and amylase activity but not trypsin or chymotrypsin activity. None of the effects suggested that this dose of TI was toxic to either the neonatal or sexually mature miniature male swine.

High-Density Er-Implanted GaN Optical Memory Devices.

Upconversion emission has been obtained from Er-focused ion-beam (FIB) implanted GaN. Visible green emission at the 522- and 546-nm range were excited with infrared (IR) laser sources at either 840 or 1000 nm, or with both lasers simultaneously. By implanting closely spaced patterns with the FIB, we demonstrated the concept of storing data in Er-implanted GaN. Information stored as data bits consists of patterns of implanted locations as logic 1 and unimplanted locations as logic 0. The photon upconversion process in Er ions is utilized to read the stored information. This process makes use of the IR lasers to excite visible emission. The integrated upconversion emission power was measured to be ~40 pW when pumped by a 840-nm laser at 265 mW and by a 1000-nm laser at 208 mW. Patterns as small as 0.5 mum were implanted and read. Three-dimensional optical memory based on rare-earth-doped semiconductors could in theory approach a storage capacity of 10(12) bits/cm(3).

Benzalkonium chloride and gentamicin cause a leak in corneal epithelial cell membrane.

The purposes of this study are to characterize the pathophysiological effects of benzalkonium chloride and gentamicin on corneal epithelial cells and to determine the concentration dependent effect of these agents on membrane currents of these cells. Rabbit corneal epithelial cells were isolated and subdivided into small, medium and large cells according to their cell capacitance. Using whole cell clamp technique, potassium current of corneal epithelial cells was recorded. Transmembrane current was measured again after bathing in benzalkonium chloride 1, 3, 10, 30 and 100 micrometer ml(-1)for 3 min. The effect of gentamicin was tested at concentrations of 0.4, 1.0, 2.0, 4.0 and 10 mg ml(-1). Synergistic effect of gentamicin of the above mentioned concentrations in the presence of benzalkonium chloride 1 microgram ml(-1)was also measured. We found that small corneal epithelial cells had the highest depolarization-gated, outward potassium current density and large cells had the lowest current density, while medium cells had a current density in between. Benzalkonium chloride induced a concentration dependent increase in the leak current with increasing concentration from 1 to 100 microgram ml(-1). The increase of leak current in medium and large cells was less than that in the small cells. Gentamicin also caused a concentration dependent increase in leak current density from 0.4 to 10.0 mg ml(-1). The increase in leak current density was statistically significant when the concentration was 2.0 mg ml(-1)or higher in small corneal epithelial cells and 1.0 mg ml(-1)or higher in the medium and large cells. Benzalkonium chloride 1.0 microgram ml(-1)augmented the effect of gentamicin on epithelial cell membrane. The extent of enhancement was more prominent in larger than smaller cells. Using whole-cell clamp technique, we were able to determine the threshold concentration of gentamicin and benzalkonium chloride on the integrity of corneal epithelial cell membrane. The toxic action of both agents is mediated by an increase in leak current. We propose that the whole-cell clamp technique is a sensitive and useful tool in determining cytotoxic effects of various agents.

[Reconstruction of laryngotracheal stenosis: experience of twenty years].

In order to improve the curative effects of laryngotracheal stenosis, 261 patients treated for laryngotracheal stenosis during the last twenty years were retrospectively reviewed. Glottic reconstruction, fence-form laryngotracheal reconstruction and laryngotracheotomy with autogenous tissue or hydroxylapatite rings were selectively used for one-stage reconstruction. Our results demonstrated that among 261 patients, 9 failed in decannulation, 5 were lost for follow-up, 247 (94.6%) patients were successfully decannulated. Among 192 patients followed-up from 1 to 18 years, 4 had restenosis 3 years after operation, 3 were successfully retreated and 10 failed. 182 (94.7%) patients had stable airways. It is concluded that there was no fixed treatment for laryngotracheal stenosis. The choice of surgical procedure and grafting must be decided on the pathologic condition of the larynx and trachea. The advantages of fence-form laryngotracheal reconstruction are simple technique and good result. Reconstruction with combined bipedicled myocutaneous flap and costal cartilage or artificial materials are better for severe laryngotracheal stenosis and large tracheal defect. The usage of stent is important for laryngotracheal reconstraction.