Although diabetes is one of the most costly and rapidly increasing serious chronic diseases worldwide, the optimal mix of strategies to reduce diabetes prevalence has not been determined.Using a dynamic model that incorporates national data on diabetes prevalence and incidence, migration, mortality rates, and intervention effectiveness, we project the effect of five hypothetical prevention policies on future US diabetes rates through 2030: 1) no diabetes prevention strategy; 2) a “high-risk” strategy, wherein adults with both impaired fasting glucose (IFG) (fasting plasma glucose of 100–124 mg/dl) and impaired glucose tolerance (IGT) (2-hour post-load glucose of 141–199 mg/dl) receive structured lifestyle intervention; 3) a “moderate-risk” strategy, wherein only adults with IFG are offered structured lifestyle intervention; 4) a “population-wide” strategy, in which the entire population is exposed to broad risk reduction policies; and 5) a “combined” strategy, involving both the moderate-risk and population-wide strategies. We assumed that the moderate- and high-risk strategies reduce the annual diabetes incidence rate in the targeted subpopulations by 12.5% through 2030 and that the population-wide approach would reduce the projected annual diabetes incidence rate by 2% in the entire US population.We project that by the year 2030, the combined strategy would prevent 4.6 million incident cases and 3.6 million prevalent cases, attenuating the increase in diabetes prevalence by 14%. The moderate-risk approach is projected to prevent 4.0 million incident cases, 3.1 million prevalent cases, attenuating the increase in prevalence by 12%. The high-risk and population approaches attenuate the projected prevalence increases by 5% and 3%, respectively. Even if the most effective strategy is implemented (the combined strategy), our projections indicate that the diabetes prevalence rate would increase by about 65% over the 23 years .While implementation of appropriate diabetes prevention strategies may slow the rate of increase of the prevalence of diabetes among US adults through 2030, the US diabetes prevalence rate is likely to increase dramatically over the next 20 years. Demand for health care services for people with diabetes complications and diabetes-related disability will continue to grow, and these services will need to be strengthened along with primary diabetes prevention efforts. International surveillance data have documented a steady rise in diabetes prevalence over the past 40 years in virtually all regions of the world ,2. AlthoWe are not aware of previous attempts to estimate the impact of different prevention strategies on future US diabetes prevalence rates. In these analyses, we used a dynamic model based on difference equations  and natiWe constructed a multistate epidemiologic model with a time-varying transition matrix that divides the US adult population into five groups: those with normal glucose levels, those with impaired fasting glucose (IFG) , those with both IFG and IGT, those with undiagnosed diabetes, and those with diagnosed diabetes. Using the model, we then estimated the impact of the following five hypothetical diabetes prevention strategies on projected diabetes prevalence rates among US adults through 2030: 1) no intervention (in which we assumed that current diabetes trends would continue unabated); 2) implementation of a national “high-risk” strategy, in which adults who have both IFG and IGT are eligible to participate in a structured lifestyle intervention; 3) implementation of a national “moderate-risk” strategy, in which adults with IFG receive a structured lifestyle intervention; 4) implementation of a “population-wide” strategy in which the entire US adult population is exposed to environmental and economic policies aimed at reducing risk factors for type 2 diabetes; and 5) implementation of a “combined” strategy, wherein population-wide strategies (strategy #4 above) are applied to the entire population while the moderate-risk strategy is simultaneously applied only to those who are identified as having IFG (strategy #3 above).We assumed that the moderate- and high-risk strategies would result in a net 12.5% reduction in diabetes incidence in the targeted groups. This is based on a scenario in which 50% of the population is identified, 50% agree to participate, and one-year incidence is reduced by 50% among those who participate -16. ThisThe principal data sources for our dynamic model were the US Census Bureau, the National Diabetes Surveillance System, and published findings from selected epidemiologic studies and meta-analyses . The modOn the basis of results of a systematic review showing annualized relative risks for diabetes associated with IFG, IGT, and IFG and IGT combined  and on oWe projected future diabetes incidence rates in the absence of any intervention using logistic growth curves and Bayesian estimation as described by Boyle et al. (2010) . PrimaryN(t) as a row vector containing the number of people in each of the five states at time t. B(t) is a row vector of births into each of the states at time t. M(t) is a row vector of net migration into each of the states. Given initial values N(2007) projections are calculated via:Our projections are based on the following five-state matrix in which we define When no intervention is applied the model is constrained to be consistent with Census Bureau projections.P(t) be a transition matrix of the probabilities of moving to or staying in each of the states at time t given the state occupied at time t-1. At any given time P(t) has the following form:We let p13 is the probability of moving to state 3 at time t given occupying state 1 at time t-1. Entries in the matrix noted 0 are transitions that cannot occur. Note that only the non-zero elements of P vary over time. The rows of P sum to one minus the death rate for the corresponding state. There were several important assumptions of the model. First, people cannot move from diabetes to no diabetes; this assumption is reasonable because remission is rare [For example,  is rare . Second, is rare .In sensitivity analyses, we also examine the impact of interventions under a scenario of background incidence of the posterior means, roughly equivalent to a continuation of the rate of increase observed over the past decade. Due to model run-time, a full Monte Carlo uncertainty assessment of uncertainty, while desirable, is infeasible. Therefore, we relied on a model emulation approach to approximate the uncertainty in the number of cases presented . DetailsIn the absence of any preventive intervention, we projected that 51.7 million new cases of diabetes among US adults would occur by 2030  would result in 4.0 million fewer new diabetes cases over 20 years, reduce the diabetes prevalence in 2030 by 3.1 million cases, and reduce the diabetes prevalence rate in 2030 from 22.7% to 21.5%. The high-risk approach (intervention for the 8.3% of the population with both IFG and IGT) would reduce the projected 2030 prevalence of diabetes by 1.2 million cases and reduce the projected prevalence rate in 2030 from 22.7% to 22.2%. The population-wide approach would have a projected impact similar to the high-risk approach, reducing 0.9 million cumulative cases, 0.7 million prevalent cases in the year 2030, and reducing the prevalence rate in 2030 from 22.7% to 22.4% , reduce the undiagnosed diabetes prevalence rate by 0.5 percentage points, but increase the predicted pre-diabetes prevalence rate by 0.9 percentage points , we fouFinally, the results of our error propagation analysis appear in Table Results of several controlled trials have shown that lifestyle interventions can reduce the incidence of type 2 diabetes among people at high risk -9,16,32.Despite the benefits that can be achieved with preventive interventions, particularly the combined and moderate-risk approaches, our projections indicate a need to develop new options for diabetes prevention and/or increase the reach of both primary and secondary prevention efforts. We projected that the diabetes prevalence rate in the United States will surpass 20% by 2030 even if the combined strategy is implemented; a large increase in prevalence will occur even at the upper credibility bound for effectiveness of this intervention, the most optimistic plausible case. This projected increase was based on data showing a steady increase in the US diabetes incidence rate over the past 20 years ,36, a deOur projection that the US diabetes prevalence rate will increase substantially even under the combined intervention scenario indicates that the success of currently known primary prevention efforts will not diminish the need for more effective programs to help people manage their diabetes and prevent its complications. Diabetes has diffuse effects, leading to multiple microvascular and macrovascular complications and an increased risk for aging-related disability. Because numerous clinical, screening, community, and educational interventions reduce the incidence of complications ,40, manyOur projections are limited by our assumptions. If the US diabetes incidence rate continues to increase at its recent pace  and all other assumptions are unchanged, the prevalence of diabetes in 2030 would be three to four percentage points higher than we projected . If mortAs indicated by both our one-variable-at-a-time and error propagation analyses, our findings are sensitive to our assumptions about the effectiveness of interventions. Results of several randomized controlled trials among people with prediabetes have found a 50% or more reduction in incidence among people with prediabetes, with some finding extended effects up to 20 years later ,16,32,43The impact of a population-wide approach to diabetes prevention is particularly hard to predict because there have been few estimates of the health impact of such an approach; this is reflected in the large uncertainty in this term in our error propagation analysis. We based our estimates that population-wide strategies would lead to a 1 to 4% reduction in the relative incidence of diabetes on the results of studies concerning community-based efforts to reduce rates of CVD ,47. GiveAlthough our projections of future diabetes prevalence rates among US adults are based on numerous assumptions, they are likely more accurate than previous projections because they are based on more recent data, including updated projections of future diabetes incidence rates and updated population projections. Most importantly, because the model we used estimated diabetes incidence and mortality rates for people at different stages of diabetes risk and in different diagnostic categories, it allowed us to more accurately project the impact of various intervention strategies. However, our projections, and others like these, need to be continuously updated as new intervention effectiveness and epidemiologic data become available. We hope that more rigorous controlled studies or perhaps natural experiments of population-targeted approaches for diabetes prevention will emerge in the future to permit more confident modeling of the impact on future prevalence.The results of our analyses indicate a need for the provision of effective lifestyle interventions to the large number of adults with prediabetes as well as support research aimed at improving the effectiveness of these interventions. They also suggest a need for research designed to improve the effectiveness of population-wide approaches to diabetes prevention. Unless both strategies are much more effective than we assumed they will be in our analyses, the prevalence of diabetes among US adults will continue to increase even if the US health care system adopts something like the combined diabetes prevention strategy that we described. This continued increase in diabetes prevalence will result in an increase in demands for diabetes management and treatment, including services related to the prevention of diabetes complications and diabetes-related disability, and these services will need to be strengthened along with primary diabetes prevention efforts.The findings and conclusions in this report are those of the authors and do not necessarily represent the official positions of the Centers for Disease Control and Prevention.The authors declare that they have no competing interests.EWG and DFW conceptualized and supervised the study and drafted the manuscript. JPB, LEB, and TJT developed the statistical models and conducted statistical analyses. ALA contributed to writing and critical review of the manuscript. All authors read and approved the final manuscript.Details of error propagation model. Table S1. Parameters used in error propogation model, including change in projected reduction in diabetes prevalence (Y), natural logarithm of magnitude of effectiveness of targeted intervention (X1), and natural logarithm of population-wide intervention effectiveness (X2). Table S2.Click here for file


Improved cancer survival poses important questions about future life conditions of the survivor. We examined the possible influence of a breast cancer diagnosis on subsequent working and marital status, sickness absence and income.We conducted a matched cohort study including 4,761 women 40–59 years of age and registered with primary breast cancer in a Swedish population-based clinical register during 1993–2003, and 2,3805 women without breast cancer. Information on socioeconomic standing was obtained from a social database 1 year prior and 3 and 5 years following the diagnosis. In Conditional Poisson Regression models, risk ratios (RRs) and 95% confidence intervals (CIs) were estimated to assess the impact of a breast cancer diagnosis.Three years after diagnosis, women who had had breast cancer more often had received sickness benefits  or disability pension  than had women without breast cancer. We found no effect on income (RR = 0.99), welfare payments (RR = 0.98), or marital status (RR = 1.02). A higher use of sickness benefits and disability pension was evident in all stages of the disease, although the difference in use of sickness benefits decreased after 5 years, whereas the difference in disability pension increased. For woman with early stage breast cancer, the sickness absence was higher following diagnosis among those with low education, who had undergone mastectomy, and had received chemo- or hormonal therapy. Neither tumour size nor presence of lymph nodes metastasis was associated with sickness absence after adjustment for treatment.Even in early stage breast cancer, a diagnosis negatively influences working capacity both 3 and 5 years after diagnosis, and it seems that the type of treatment received had the largest impact. A greater focus needs to be put on rehabilitation of breast cancer patients, work-place adaptations and research on long-term sequelae of treatment. Cancer survival has improved during the last decades, particularly for patients diagnosed with breast cancerIn the present study we used information retrieved through record linkage between three population-based registries to investigate possible socioeconomic consequences of breast cancer survivors in terms of sickness benefits, marital status, welfare dependence, and income changes 3 and 5 years following a breast cancer diagnosis. The investigation was made possible by comparing breast cancer patients with women without breast cancer randomly selected from the same population. We also investigated the possible influence of tumour characteristics and type of cancer treatment on the likelihood of returning to work.This study was based on a dataset generated by record linkage between three population-based registers, the Regional Quality Register of Breast Cancer of the Uppsala/Örebro Region in Central Sweden, the National Population Register, and the LISA-database (an integrated database for labour market research). Linkage was made possible by the individually unique National registration number assigned to each resident in Sweden at birth or time of permanent residency.The Regional Quality Register of Breast Cancer in the Uppsala/Örebro health care region covers seven counties in central Sweden with a source population of 1.9 million . The main purpose of the register is to monitor the quality of care based on regional or national guidelines for breast cancer management. The register includes individual information reported continuously from the clinicians on date of diagnosis, detection mode, tumour-stage, tumour characteristics and primary surgical and oncological treatment for all newly diagnosed breast cancer patients. The database is continuously updated against the National Population Register to assess current vital status of the registered patients. The Regional Quality Register of Breast Cancer was started in 1992 and has been estimated to have a coverage of 97%, following a validation against the records of the mandatory Swedish National Cancer RegisterThis register, which is the basic register of the population of Sweden, contains information about who lives in Sweden and place of residence. The register is managed by the Swedish Tax AgencyIndividual information on socioeconomic and demographic factors was obtained from the LISA-database managed by Statistics SwedenWe investigated patients reported to the Uppsala/Örebro Regional Quality Register of Breast Cancer between January 1, 1993 and December 31, 2003.Information from LISA was collected for the years 1992  to 2006. This means that we had a follow-up of 5 years for all women diagnosed between 1993 and 2001, but a follow-up limited to 3 years for women diagnosed between 2002 and 2003 .This group was defined as all women aged 20–59 years with a diagnosis of primary invasive breast cancer between January 1, 1993 and December 31, 2003 and reported to the Uppsala/Örebro Regional Quality Register of Breast Cancer. Since we were interested to investigate the consequences of cancer on those who we were able to follow for 5 years or more (or 3 years for women diagnosed between 2002 and 2003), women with incomplete follow-up were excluded. In the initial database of 5482 patients, 4413 were diagnosed between 1993 and 2001, and 1069 women between 2002 and 2003. Of the 4413 women diagnosed between 1993 and 2001, 3776 (85.6%) could be followed for 5 years, and of the 1069 women diagnosed between 2002 and 2003, 986 (92.2%) could be followed for 3 years. This means that from the initially identified 5482 patients, 721 were lost to follow-up and therefore excluded. The main reason for failure to follow-up was death, i.e. 94.8% (676 of 721) of the women had died within the follow-up period. The other main reason for lost follow-up was that the women had moved out of the health care region. Proportionally more women with advanced stages (III-IV) were lost to follow-up than were women in earlier stages, i.e. of the original sample 89% (4095/4587) of women with stages I-IIB could be followed compared to 64% (225/354) of the women with stages III-IV. Also, the women lost to follow-up were proportionally younger, i.e. of women younger than 44 years of age 83% could be followed (898/1080 = 83%) compared to 88% (3863/4394) of women aged 45-59 years (data not shown). In one prior study we also found that younger women had a poorer 5 year relative survival after breast cancerEach patient was individually matched to 5 women without breast cancer  registered in the National Population Register with complete follow-up by birth year, and community one year prior to the breast cancer patients' calendar year of diagnosis. The main reason for matching was to minimise confounding that may influence the outcome of investigated variables, such as working status and income. The matching was conducted by Statistics Sweden, and made possible by use of the National registration number  and information available in the National Population Register .We retrieved information on patients with early stage breast cancer classified according to tumour stage , tumour-size in millimetre , lymph node involvement , primary surgical treatment , and intended oncological treatment . We also investigated the effect of sentinel node biopsy and axillary dissection . Since the sentinel node procedure not was fully introduced in our health care region until 2005, only few node-negative patients were staged with this method in the study cohort leading to low power and making it difficult to generalize results.From this register we retrieved individual information on all women 1 calendar year prior to the breast cancer patients diagnosis as well as 3 and 5 years after the year of diagnosis on education , working at least part-time , unemployment , retirement , use of welfare , marital status  and number of people in the household .Sick pay can be obtained (from the employer) the first 14 days of a sick period. Sickness benefit is paid by the Swedish Social Insurance Agency if the employee is ill for a longer period than 14 days. The sickness benefit is approximately 80% of the individuals income up to a certain limit (a year wage of SEK 321 000). Sickness pension (or disability compensation) can be received if the work capacity is permanently reduced by at least a quarter. Full income-related sickness compensation provides 64% of the individuals assumed income. Sickness absence in our study was defined as all women registered during the year of follow-up with sickness benefit or disability pension at least part time . We also presented the values of sickness benefit , and disability pension .The social insurance system in Sweden covers everyone that resides or works in Sweden, and provides financial protection for persons with a disability or in connection with an illness. The disposable income is the individual's total income  minus taxation. To show possible differences in income 1 year prior to diagnosis between women with and without breast cancer we categorized income as lowest 25%, 25–50%, 50–75% and 75–100% . To inveDue to the social insurance system in Sweden (see above) it is difficult to use either employment or income as an outcome variable. A person could be registered as working full-time if he/she is on sick-leave up to 100% from full-time work. Also, the disposable income (up to a certain income level as mentioned above) is not affected to a great extent. We therefore chose to use sickness absence as our main outcome variable.In order to compare women diagnosed with breast cancer with their matched controls 1 year prior to diagnosis, percentages in various categories of the following variables were compared: education, working at least part time, sickness absence, unemployment, retirement, welfare, disposable income, marital status, and number of people living in the household. To quantify the differences between the two groups conditional logistic regression was used to account for the matching with differences reported as odds ratios (OR) with 95% confidence intervals (CI). Only unadjusted estimates are reported, as the main interest was to investigate whether there were any imbalances between the groups before the women with breast cancer had been diagnosed .In order to investigate differences in working life, income and marital status in women diagnosed with breast cancer with their matched controls at 3 and 5 years post diagnosis, conditional Poisson regression was used to estimate a risk ratio (RR) with a 95% CI. A separate model was fitted for each factor of interest with each model adjusted for education level and the factor of interest recorded 1 year prior to diagnosis .In order to compare the effect of stage at diagnosis and treatment on sickness absence at 3 and 5 years post diagnosis for women diagnosed with breast cancer, unconditional logistic regression was used with differences reported as odds ratios with 95% CIs. The following factors were investigated, working 1 year before diagnosis, education, tumour size, having lymph nodes, sentinel node, stage, and treatment. For each variable two models were compared; the first model adjusted for education and the factor of interest one year prior to diagnosis and the second model adjusted for education and the factor of interest one year prior to diagnosis and the remaining investigated factors .Compared to women with low education, the risk of breast cancer was higher for women with high  and middle  education. Also, the risk of a breast cancer diagnosis was somewhat higher among women who worked , and lower among women who had received welfare allowance  . No signBreast cancer had a post diagnostic effect both 3 and 5 years after the diagnosis on sickness benefits and disability pension . In the The proportion of women who had received sickness benefit was after 3 years 19.2% among women without breast cancer, compared to 25.3%, 33.0%, and 38.2% in women with breast cancer stage I, II, III-IV, respectively and after 5 years 19.7% among women without breast cancer, compared to 23.9%, 26.2%, and 22.1% in women with breast cancer stage I, II, III-IV, respectively.The proportion of women with disability pension was after 3 years 19.3% among women without breast cancer, compared to 22.6%, 26.1%, and 28.0% in women with breast cancer stage I, II, III-IV respectively, and after 5 years 21.7% among women without breast cancer, compared to 26.1%, 32.5%, and 34.3% in women with breast cancer stage I, II, III-IV, respectively .After adjustment for sickness absence and education 1 year prior to the diagnosis, the likelihood for women with stage I-IIb disease to be on sick leave was positively associated with larger tumours, lymph node metastasis, treatment by mastectomy and receiving adjuvant chemo- or hormonal therapy, whereas treatment by radiotherapy had no effect Model 1, . After aThe effects of tumour characteristics and treatment were somewhat weaker compared to those after 3 years except that of education. The likelihood of women with stage I-IIb disease to be on sickness absence was positively associated with a low educational level, large tumours, lymph node metastasis and treatment by mastectomy, chemotherapy or hormonal therapy, whereas treatment by radiotherapy had no effect Model 3, . After achemotherapy increased the risk for sickness benefit in the 3'rd but not in the 5'th year after diagnosis , but had no effect on the risk for disability pension either 3 or 5 years after diagnosis . Hormonal treatment also increased the risk for sickness benefit 3 but not 5 years after diagnosis , but did, in contrast to chemotherapy, have an effect on the risk for disability pension both 3 or 5 years after diagnosis  (data not shown).After adjustment for tumour characteristics, treatment by Sentinel node: The risk of sickness absence was higher among patients with stage I-IIb breast cancer who had had axillary lymph node dissection compared to sentinel node biopsy  Contrary to findings in earlier studiesIn contrast to other investigationsThe major determinants for sickness absence after 5 years were level of education and type of treatment. Low education, low socioeconomic standing and having a manual job have previously been reported to be risk factors for early retirement and unemployment after a cancer diagnosisTreatment by mastectomy and axillary dissection were also risk factors for sickness absence after 3 and 5 years. Mastectomy has also been reported as being associated with an increased risk for chronic painAdjuvant chemotherapy, did however, negatively affect both sickness benefit and disability pension after 3 years, but not after 5 years. This finding corroborates results from other studies reporting short time effects of chemotherapyAdjuvant hormonal therapy did have a persistent impact on disability pension even after 5 years, which is in line with prior investigations associated with returning to work Even in early stage breast cancer, the diagnosis was negatively associated with sickness absence both 3 and 5 years after diagnosis, and was most pronounced in women who underwent mastectomy or received chemotherapy or hormonal treatment. The knowledge about how to increase the number of women returning to full-time work is rather unexplored, and a study has also found that many cancer patients report unmet rehabilitation needs


Diabetes mellitus is a risk factor for cardiovascular disease; however, the mechanisms through which diabetes impairs homeostasis of the vasculature have not been completely elucidated. The endothelium interacts with circulating blood through the surface glycocalyx layer, which serves as a mechanosensor/transducer of fluid shear forces leading to biomolecular responses. Atherosclerosis localizes typically in regions of low or disturbed shear stress, but in diabetics, the distribution is more diffuse, suggesting that there is a fundamental difference in the way cells sense shear forces. In the present study, we examined the effect of hyperglycemia on mechanotranduction in bovine aortic endothelial cells (BAEC). After six days in high glucose media, we observed a decrease in heparan sulfate content coincident with a significant attenuation of the shear-induced hydraulic conductivity response, lower activation of eNOS after exposure to shear, and reduced cell alignment with shear stress. These studies are consistent with a diabetes-induced change to the glycocalyx altering endothelial response to shear stress that could affect the distribution of atherosclerotic plaques. Hyperglycemia is a characteristic feature of type 1 and type 2 diabetes that has been long associated with both micro and macrovascular dysfunction 2) and vasoconstrictors such as endothelin (ET) The interaction between blood flow and the endothelium is not only a result of soluble factors, but also includes the mechanosensing of shear forces that are exerted by blood flow The GCX, which covers the surface of the endothelial cells, consists of proteoglycans linked either to the membrane (glypicans) or to the cytoskeleton (syndecans) with extracellular side chain glycosaminoglycans (GAGs) covalently linked to the core proteins. The most abundant of these GAGs are heparan sulfate (HS), chondroitin sulfate (CS) and hyaluronic acid (HA) The GCX may contribute as much as 60% of the hydraulic resistance of the capillary wall Zuurbier et al., The above studies provide evidence to support the hypothesis that hyperglycemia interferes with either the synthesis and or the shedding of at least two GAGs – HS and HA. However to our knowledge, there have been no studies that address whether hyperglycemia impairs the shear-induced Lp response. Previous in-vitro studies have established that shear stress increases endothelial hydraulic conductivity and that this effect can be attenuated by an eNOS inhibitor The following chemicals were obtained from Sigma-Aldrich Chemical Company : bovine serum albumin (BSA-30% solution), Minimum Essential Medium Eagle (MEM), phenol red-free MEM (PF), penicillin-streptomycin solution, L-glutamine, trypsin-EDTA solution, HEPES, sodium bicarbonate, heparin , fibronectin, and (6R)-5,6,7,8-Tetrahydrobiopterin (BH4). Fetal bovine serum (FBS) was purchased from Hyclone Laboratories . Transwell polyester filters  were purchased from Costar . Primary Anti- Heparan Sulfate 370255-1 was purchased from Amsbio , Biotinylated Hyaluronic Binding Protein 385911 was purchased from Calbiochem  and primary Anti-Glypican-1 66909 and Anti Syndecan-1 5632 were purchased from Santa Cruz . The secondary antibodies IgM and IgG were purchased from Molecular probes  and the Western blot kit, from Cayman . The Phospho-eNOS (Ser1177) and β-actin antibodies were obtained from Cell Signaling .2 balanced air. The model for the endothelium was developed by plating BAECs at a density of 6×104 cells/cm2 on 12 and 24 mm diameter Transwell polyester filters previously coated with fibronectin. The filters were incubated for 6 days with either 5 mM glucose (NG) or 25 mM glucose (HG) 10%FBS-MEM. Cells were used from passages 5 to 10.BAEC were purchased from VEC Technologies  and from Lonza  and grown in T-75 flasks with 10% FBS-MEM. The flasks were kept at 37°C in 5% COAn apparatus developed in our lab 2O differential pressure was applied, the volumetric flow rate (Jv) was measured by tracking the position of a bubble that was inserted into the calibrated borosilicate glass tube. The hydraulic conductivity was calculated from the relationship: Lp  =  (Jv/A)/ΔP, where A is the BAEC monolayer area and ΔP is the pressure differential across the monolayer. After 60 minutes of applied pressure differential to drive water flux, a baseline Lp was established, and a defined shear stress was applied to the endothelial monolayer using a rotating disk separated by a distance h (500 µm) from the monolayer surface. The rotating disk generated a fluid shear stress distribution on the monolayer surface defined by: τ = μ×ω×r/h, where μ is the viscosity of the media, ω is the rotational speed, and r is the radial distance from the center of the disk. The parameters were adjusted in order to achieve a maximum steady shear stress of 20 dyn/cm2 at the edge of the disk, and this is the value that is reported in the tables and figures. The average shear stress over the entire filter area is 2/3 of the maximum. During the experiment, the luminal compartment and the reservoir were supplied with gas  to maintain the experimental media at the physiological pH of 7.4. The Lp values were recorded for a total of four hours.The seeded 12 mm filters were placed inside a chamber to form a luminal (top) compartment and an abluminal (bottom) compartment separated only by the BAEC monolayer. The abluminal compartment was connected to a reservoir via Tygon and borosilicate glass tubing. The vertical displacement of the reservoir with respect to the liquid covering the cells, allowed us to apply a hydrostatic pressure differential across the monolayer. When a 10 cm HTwo experiments were always run side-by-side. One endothelial monolayer was grown in normal glucose, while the companion monolayer was grown in high glucose. These pairs of monolayers originated from the same flask and passage and were grown for six days in the same tray.Monolayers were grown in either NG or HG for six days. For immunocytochemistry, cells were rinsed with cold PBS and fixed with 4% paraformaldehyde-PBS for 15 minutes, blocked with 4%BSA-PBS for one hour, incubated with either of the following primary antibodies: Heparan Sulfate, Hyaluronic Acid, Syndecan-1 and Glypican-1diluted 1∶50 in 4% BSA-PBS for 3 days and washed gently with 1% BSA-PBS. The monolayers were incubated with secondary antibody for 1 hour: Alexa Fluor 488 conjugated with anti-mouse IgM or anti-biotin or anti-rabbit IgG diluted 1∶500. The monolayers then were washed 5 times and at least 4 random fluorescent images where taken per condition with the LSM 510 microscope manufactured by Carl Zeiss Inc. The intensity of the images was quantified with the software image J.BAEC monolayers were grown on 24 mm diameter inserts for six days in either NG or HG, then the media was replaced with 5% FBS- 5%BSA MEM and the inserts were placed inside the compartment of an apparatus that was designed to hold a 6 well tray with inserts. A rotating disk above each insert generated a fluid shear stress distribution on the monolayer surface as described above. All 6 disks with shafts were driven by a humidity resistant stepper motor and controller . The apparatus holding the inserts was placed inside the incubator to control for temperature and pH, and the monolayers were sheared for 24 hours at a maximum level of 12 dyne/cm2. At the end of the experiments the inserts were removed and phase contrast photographs taken at randomly selected locations (n = 4) using the Nikon Eclipse TE2000-E inverted microscope. These fields were all located toward the outer edge of the rotating disk where the shear stress was maximal. The length, width and angle of alignment of the cells in the field were measured with image J. The results were reported as the aspect ratio and alignment angle. A total of 24 fields were chosen for each condition (HG vs. NG).BAEC monolayers grown in either NG or HG were placed inside the 6 well shearing apparatus where they were sheared for 4 hours at a maximum level of 20 dyne/cm2. During this time, the apparatus with the cells as well as the static controls were maintained inside the incubator to control for temperature and pH. Then the cells were collected for western blots to quantify total and phosphorylated eNOS.The activation of eNOS was determined after shear stress was applied for 4 hours, the monolayers were washed with cold PBS and scraped from the filters with a plastic spatula in the presence of RIPA extraction buffer . Protein concentration was determined with the DCTM Protein Assay from BioRad  using the ND2000 spectrophotometer from NanoDrop . Western blot was carried out by standard techniques, loading 20 μg of protein into the gel wells and incubating over night with antibodies to eNOS and phospho-eNOS (Serine 1177), and the constitutively expressed protein β-actin, all from Cell Signaling Technologies , followed by reaction with matched secondary HRP conjugated anti-rabbit and anti-mouse IgG antibodies from Molecular Probes . The blots were scanned with the System FX Pro imaging system from Carestream  and quantified with Image J software. The same general procedure was carried out to collect protein from static monolayers treated with normal and high glucose as well as Mannitol controls, placing 30 ug of protein into the gel wells, and incubating with primary antibodies for Syndecan-1 and Glypican-1 diluted at a ratio of 1∶500.P<0.05 was considered significant.Lp values, Heparan Sulfate, Hyaluronic Acid, Syndecan-1, Glypican-1 fluorescence, eNOS, Syndecan-1, Glypican-1 protein expression and alignment measurements are presented as means ± SE. Tests for statistical significance of Lp values were conducted using a 2-way ANOVA function from Excel, and a post hoc analysis using the Tukey method. Western blots, immunostaining and alignment pictures were quantified and analyzed using the NIH image J software. In these cases, statistical significance was determined using a 2-tailed t-test. For all of the experiments, 2O pressure differential) for normal vs. high glucose were not significantly different from one another . Both values of the baseline Lp were within the normal ranges that have been reported for BAECs in previous studies High glucose did not affect the “sealing effect” but significantly attenuated the shear induced Lp response. A similar behavior was observed for monolayers treated with heparinase The visualization of Heparan Sulfate, Hyaluronic Acid, Syndecan-1 and Glypican-1 in the GCX was performed carefully, and it was necessary to fix the cells to preserve structural integrity. As shown in For the Western blots the β-actin band served as a loading control and the ratio of p-eNOS to eNOS was obtained after normalizing the levels of each lane to each corresponding β-actin control. As shown in Additionally, the blots were re-probed with an antibody for total eNOS to make certain that a difference in phosphorylation was not a result of a change in the total eNOS protein. The total amount of eNOS protein did not differ between monolayers cultured in normal vs. high glucose .Representative images of the alignment experiment are shown in The observation that with diabetes atherosclerosis is distributed more uniformly in arteries, and not just limited to the typical atherogenic regions (regions of low shear or disturbed flow) near bifurcations and curvatures To that end, we designed a study to determine if high glucose impairs the endothelial response to shear stress. Shear stress has been shown to regulate endothelial hydraulic conductivity by a mechanism that involves the transduction of mechanical forces into the production of NO The main observations of the present study were that the normal Lp increase caused by a step change in steady shear stress was significantly attenuated by high glucose , coincidPrevious studies have linked high glucose to dysfunction of the endothelium The GCX has received a great deal of attention for its role in mechanotransduction in recent years. Florian et al. The downregulation/degradation of HS by hyperglycemia could have important implications for diabetes. Several atheroprotective properties have been attributed to HS proteoglycans based on their capacity to inhibit monocyte binding to the subendothelial region In human arteries, hyperlipidemia has been characterized by decreased HS In other tissues, such as kidney With respect to mechanosensing and adaptation, another in-vitro study showed that endothelial monolayers cultured in high glucose had reduced HS and were not able to align normally after being exposed to shear stress The shear induced Lp response, which is mediated by the production of NO Other studies have shown that phosphorylation of eNOS by several serine/threonine kinases is a fundamental step in NO production by endothelial cells. Phosphorylation by AMP kinase, Akt, (protein kinase A), or protein kinase B, on serine 1177, leads to increased activation of the enzyme and increased NO production in bovine cells Another possibility for a reduced endothelial response is that once released, NO is quickly modified and its potency is reduced in a hyperglycemic environment due to the presence of advanced glycation end products which also modify eNOS Endothelial dysfunction (ED) explains an increased risk for cardiovascular disease in diabetic patients. Improved understanding of the mechanisms that lead to ED in this setting could provide new approaches for treatments. The mechanisms through which hyperglycemia causes ED are varied and may present complex interactions, however, the findings in this study point to the GCX as a possible target for early therapeutic intervention. A future approach for vascular disease may be based on the design and administration of copolymers


Accurate prediction of the biochemical recurrence (BCR) is critical for patients after intended curative therapy like radical prostatectomy (RP) or definitive radiotherapy for prostate cancer. Glutathione-S-transferases polymorphisms as well as hypermethylation of GSTP1 and functional genes in carcinogenesis, including tumor suppression gene (APC), hormone receptor that regulates cell growth and differentiation gene (RARbeta) were reported to be associated with BCR. Nevertheless, the reported results are inconsistent. To evaluate the relationship between glutathione-S-transferases polymorphisms and hypermethylation of these genes and the risk of prostate cancer BCR, we carried out a meta-analysis of the published studies.We performed a search in Medline, Embase and CNKI database with GST, APC, RARbeta in combination with single nucleotide polymorphism, hypermethylation, prostate cancer and recurrence. Languages were restricted to English and Chinese.Our study included 4 case-control studies and 7 cohort studies including 12 data sets and 3,037 prostate cancer patients. We confirmed that APC hypermethylation is associated with a modest hazard for biochemical recurrence after RP . We also suggest GSTP1 polymorphism and CpG hypermethylation tested in serum are associated with BCR . We also identified a possible association between GSTM1 null polymorphism and prostate cancer biochemical recurrence risk with borderline significance .To our knowledge, this is the first meta-analysis evaluating the relationship of polymorphisms and hypermethylation in GSTs and biochemical recurrence. GSTM1, GSTP1 polymorphisms and hypermethylation of GSTP1, APC may be potential biomarkers for the evaluation of the probability of BCR. Further studies are warranted to validate these findings in larger cohorts with longer follow-up. Prostate cancer (PCa) is the most commonly diagnosed cancer and the second leading cause of cancer-related deaths for men in the western world Several nomograms have been developed to predict subsequent risk of BCR after RP. They generally rely on known clinical and pathologic variables including PSA, Gleason score, clinical stage, and the number of positive and negative biopsy cores The glutathione-S-transferases (GSTs) are phase II enzymes involved in detoxification of reactive oxygen species and environmental carcinogens, metabolism of steroid hormones and chemotherapeutic agents Epigenetic are changes in gene expression not caused by alterations in the primary sequence of the nucleotides of the gene. DNA hypermethylation is the most common epigenetic change and one of the most common molecular alterations in human cancer This work was approved by the Institutional Review Board of Changhai Hospital and was performed in accordance with the PRISMA 2009 Checklist for the conduct of meta-analysis . We carrStudies included in the meta-analysis must meet all of the following criteria: (a) evaluation of the glutathione-S-transferases polymorphisms, CpG hypermethylation and prostate cancer recurrence, (b) using a cohort or case-control design, (c) using a Cox proportional hazards modeling, (d) sufficient published data for estimating an hazard ratio (HR) with 95% confidence interval (CI), and (e) article was either in English or Chinese. Accordingly, the exclusion criteria were: (a) reviews and repeated literatures, (b) not offering the source of cases and controls and other essential information, and (c) not designed as case control or cohort studies. If studies had proper design for this meta-analysis but did not have enough data, an email was sent to the authors for further supplementary data Data were independently abstracted by two investigators (RC and TM) using a standard protocol and data-collection form in accordance to the criteria stated above. Differences among evaluators were resolved by discussion and rereading with the third investigator (SR). The following information was extracted from each included study using a standardized data collection protocol , Table 2Most studies on GSTs polymorphisms uses multivariable Cox proportional hazards model. So we extract HR and 95%CI in the multivariable model that were adjusted for age, Gleason score, tumor stage at diagnosis, PSA screening history, smoking, and radical prostatectomy status as the multivariable model listed in The strength of the association between these polymorphisms or hypermethylation of promoter region and time to PCa biochemical recurrence was measured by HRs with 95% CIs. Odds ratios (ORs) or relative risks (RRs) measure only the number of events and take no account of when they occur are appropriate for measuring dichotomous outcomes, but less appropriate for analyzing time-to-event outcomes. The statistical significance of the summarized HR was determined by the Z-test. For GSTM1 and GSTT1 null polymorphisms, we estimated the impact of the ‘‘Null’’ genotype on time to recurrence, compared with the ‘‘Present’’ genotype. When it comes to GSTP1, the risk of biochemical recurrence evaluated is GSTP1 “AG vs. AA” and “GG vs. AA”, respectively. We didn’t evaluate other models (e.g. GG and AG vs. AA) because the data from included studies provides not sufficient data. As for hypermethylation of GSTP1 and other genes included, the risk of “promoter hypermethylation” compared with “not hypermethylation” was estimated. Homogeneity was evaluated by x2-based Q-test. If this test is rejected using a p-value cut point of 0.10 or less then there is sufficient evidence for the existence of heterogeneity and a lack of homogeneity. In this situation we utilize a random-effects model (the DerSimonian and Laird method) All potentially eligible studies investigating the relationship between GST polymorphisms or the methylation of promoter region and prostate cancer recurrence were identified. During the extraction of data, 12 articles were excluded, because they did not provide essential data leaving 11 eligible articles including 12 data sets involving 3,037 prostate cancer patients There was significant heterogeneity across the studies of the GSTT1 null polymorphism, GSTP1 AG vs. AA polymorphism and GSTP1 hypermethylation. So a random-effect model was utilized to analyze these data and the source of heterogeneity was further explored in the sensitivity test. For other meta-analysis, the fixed-effect model was implied and the results of random-effect model have been compared as a type of sensitivity test.For GSTM1 null polymorphism, none of the four included studies suggested a significant association with biochemical recurrence of prostate cancer. However, the meta-analysis in the fix model indicates that this polymorphism is associated with a 1.3-fold risk for biochemical recurrence with borderline significance  . So we mh = 0.08, I2 = 0.57). The results indicate GSTT1 null polymorphism to be a modest risk factor for biochemical recurrence  . In the APC hypermethylation was associated with an increase risk of prostate cancer biochemical recurrence  , Table 4Begg’s funnel plot and Egger’s test were performed to assess the publication bias of the currently available literature. The shapes of the funnel plot for the comparison of all the gene polymorphisms and promoter hypermethylations appeared symmetrical. Egger’s test was used to provide statistical evidence for funnel plot symmetry. The p-values of the Egger’s tests are 0.55 for GSTM1, 0.78 for SGTT1, 0.47 for GSTP1 AG vs. AA and 0.60 for GSTP1 GG vs. AA The results did not suggest any evidence of publication bias .For all gene variants, sensitivity analysis was performed by excluding one or more studies at one time. We estimated the summarized effect in stratified analysis by race, sample type or method to test methylation. Results of stratified analysis have been listed in For GSTM1 null polymorphism, when we perform stratified analysis in studies in Caucasian, the summarized results indicated a moderate hazard for GSTM1 null polymorphism at a borderline significance . As for GSTT1 null polymorphism, results of any stratification failed to yield a significant association with prostate cancer BCR. GSTP1 GG polymorphism showed a 1.5-fold risk of BCR over GSTP1 AA when the analysis is confined to studies using serum sample . Subgroup analysis of studies in Caucasians has achieved similar results with a lower HR and a borderline significance similar with results of the overall analysis . GSTP1 hypermethylation status was not significant associated with BCR in the overall analysis, however, when we confine the analysis to those carried out using serum samples with restriction endonuclease quantitative PCR to test methylation level, hypermethylation act as a significant risk factor for BCR . When we use the random-effect model for those studies with a low heterogeneity, the results are quite close to the data we got from the fixed-effect model . Thus weIn current standard of care, biochemical recurrence after RP serves as a trigger point for further treatment; therefore any biomarker that is correlated with biochemical recurrence would be a valuable tool for the clinical management of the disease. Polymorphism of GSTs has been extensively studied unveiling a possible association with prostate cancer susceptibility and risk of biochemical recurrence. This meta-analysis supports the association with GSTM1 and GSTP1 polymorphism with an increased risk of BCR with borderline significance. The proteins GSTM1 and GSTP1 encode are known to have an important impact in modification of some enzymes. These enzymes may have function in the detoxification of electrophilic compounds, including carcinogens, therapeutic drugs, environmental toxins and products of oxidative stress, by conjugation with glutathione We put special emphasis on GSTP1, because its methylation has been shown to occur early in high-grade prostatic intraepithelial neoplasia (HGPIN), suggesting the possibility of using GSTP1 to detect very early stage of recurrence In this meta-analysis, we suggest APC hypermethylation can pose a modest hazard for BCR after RP. The results also indicated that GSTP1 hypermethylation tested in serum may be an effective indicator for BCR after RP. GSTP1 GG polymorphism tested in serum has been illustrated to poses a hazard to BCR in overall population with a borderline significance and significant results were yield in the studies using serum as test sample. Taking the limited sample size of included studies, we may believe that if more cases are enrolled this effect may be more significant.In the 11 studies included, 4 are designed as case-control studies and 7 are cohort studies. All the case-control studies have selected the proper controls and the cohort studies are also well-designed. Furthermore, nine studies indicated a median time follow-up ranging from 1.7 to 9 years and two studies failed to offer the median time follow-up. However, the median time to BCR after RP ranged from 1.7 to 8 years and in 7 studies this data was not reported, suggesting the possibility of insufficient follow-up. In the publication search period we excluded articles in languages other than English and Chinese. When considering studies of other language, only one study in German was qualified; however, there was inadequate data in the abstract to be included.In interpreting the results, some cautions should be applied. First, the heterogeneity and small sample size may have distorted this meta-analysis. For instance, a few published studies lacked the essential data required and not all the articles set RP as an initial treatment, making the effects of different treatments on the time to BCR and rate of BCR unclear. Further studies should discriminate between various treatments and focus on the BCR after a single therapeutic modality such as RP. Similarly, there is some heterogeneity in the aspect of ethnicity, as some of the studies investigated mixed population. Secondly, currently available studies failed to investigate the relationship between race and gene polymorphisms and methylation. Subsequent studies should concentrate on exploring the genetic and epigenetic differences that exist among the different races. Thirdly, although available genetic data suggest an increased risk for BCR with APC, GSTM1 and GSTP1 promoter methylation, we still lack the knowledge of their gene-environment interactions. Further studies are warranted to confirm these findings.In conclusion, to our knowledge this is the first meta-analysis evaluating the polymorphisms and methylation in GSTs and biochemical recurrence. We confirmed that APC CpG hypermethylation poses a modest hazard for BCR after RP. We also suggest GSTP1 polymorphism and CpG hypermethylation tested in serum are probably associated with BCR. There are the potential implications of these SNPs and epigenetic change for evaluation of the probability of BCR. Further studies are warranted to validate these findings in a larger cohort with a longer follow-up.Figure S1Funnel plot of publication bias.(DOCX)Click here for additional data file.Checklist S1PRISMA 2009 Checklist.(DOC)Click here for additional data file.


Onchocerca volvulus (Ov). An estimated 180 million people are at risk for Ov infection, and 37 million people are infected, mostly in Africa. A lateral flow-based assay to detect human IgG4 antibodies to the Ov-specific antigen Ov-16 was developed as a rapid tool to detect exposure to Ov. The test, when performed on 449 sera specimens from patients with microfiladermia and Ov-negative patients, has a sensitivity of 89.1% , and specificity of 97% . Because the intended use of the test is for surveillance, it is highly desirable to have a stable, long-lasting result. An extended read window is thus desirable for a high-volume, busy workflow and facilitates post-surveillance quality assurance. The main restriction on achieving an extended read window for this assay was the erythrocyte lysis that can alter the signal-to-noise ratio, especially in those with low IgG4 levels (weak positives). We describe a test housing that incorporates a user-independent feature driven by assay fluid and an expanding wick that detaches the blood separation membrane from the nitrocellulose used in the assay, but before hemolysis occurs. We demonstrated material functionality at extreme operational conditions  and a read window of a minimum of 70 days. The fluid-driven assay device performs equally as well with whole blood as with plasma, as demonstrated with 100 spiked clinical specimens (with a correlation coefficient of 0.96). We show a novel, inexpensive, and simple approach to actuating the detachment of the blood separation membrane from the nitrocellulose test with no impact on the performance characteristics of the test.Onchocerciasis is a neglected tropical disease caused by infection with the parasite  Onchocerca volvulus (Ov). The disease affects approximately 37 million people in Africa and the Americas, with over 500,000 people visually impaired and 250,000 people blinded by the disease Onchocerciasis, or “river blindness,” is a treatable neglected tropical disease caused by infection with the parasitic helminth Current tests for the definitive diagnosis of infection with Ov involve identification of subcutaneous nodules or direct observation of the Ov microfilariae by skin snip and microscopy. Skin snips combined with microscopy are the gold standard, but are relatively insensitive when microfilarial (MF) skin densities are low. Polymerase chain reaction of the skin snips using the O-150 repeat sequence as the target provides significantly greater sensitivity r = 0.815; P<0.038). In Côte d’Ivoire, the Ov-16 card test had 76%–81% sensitivity  in communities without a history of ivermectin for vector control measures An alternative approach to the identification of incident infections in communities having already undergone mass drug administration involves using antibody detection to Ov-specific antigens that are expressed by the larval stages (L3 and L4) of the parasite. Several Ov-specific antigens have been assessed in the past PATH, in collaboration with the National Institute of Allergy and Infectious Diseases, has been developing a lateral flow test for technology transfer to a manufacturer and commercialization. One of the key design input features is the stability of the test results output such that a reading 20 minutes after the test is performed gives the same result as a reading 24 hours or even weeks later. This allows for quality assurance of tests performed in the field as well as alleviating a time constraint in a busy work flow that is typical for staff members performing large-scale epidemiological assessments.The Ov-16 RDT, as with many point-of-care lateral flow serology tests, requires red blood cell separation. The need to separate serum from platelets, white blood cells, and/or red blood cells may vary depending on the application, but is generally for reasons of chemical or visual separation. Red blood cells and their lysed fragments can visually obscure the test and/or control lines, resulting in invalid or false results and can even lead to costly product recalls. One of the device parameters for the Ov-16 RDT determined in the user-centric design process is to have test results that are stable from 20 minutes to 30 days. To achieve this using a finger-stick whole blood sample, the device must effectively separate red blood cells from serum and prevent them and their lysed fragments from entering into the reaction zone. Currently available products for separating red blood cells from serum used in manufacturing lateral flow tests fail with increased sample volume or release lysed fragments over time.Here we demonstrate a housing strategy that ensures the stability of test results well beyond 30 days. The technology involves an expandable material to separate the blood separation membrane from the nitrocellulose test strip in an Ov-16 RDT after the sera has entered the test but before hemolysis of the red blood cells begins to occur. We demonstrate the performance of an Ov-16 RDT incorporated with this technology on 100 clinical specimens and compare the performance against microfilaria status and Ov-16 IgG4 status as determined by ELISA.Onchocerca volvulus MF-positive sera and 50 Onchocerca volvulus-negative sera. The spiked samples were used fresh within 8 hours of preparation.All samples used were acquired under a number of registered protocols that were approved by the Institutional Review Board of NIAID with the majority being collected under NCT00001230. Written informed consent was obtained from all subjects prior to collection of the samples, and all the subjects consented to having serum stored for later analysis. Samples were either of serum or plasma and had been stored at −80°C until use. A positive specimen pool was made by combining four larger-volume specimens positive for IgG4 antibodies to Ov-16 antibody. The Ov-16 assay, both on the lateral flow test and on the ELISA, was evaluated on a panel of 449 sera specimens of known MF status see . From thE.coli and purification on glutathione-agarose columns (Thermofisher) as has been described previously Recombinant Ov-16 glutathione-S-transferase fusion protein was prepared through expression of the protein from a pGEX vector in Ov-16 antigen is adsorbed to Immulon 2HB plates in 1× PBS, pH 7.4, at 5 µg/ml concentration overnight at 4°C. A solution of 1× PBS +0.05% Tween 20 and 5% fetal bovine serum (PBST+5% FBS) is used as both the blocking solution and assay diluent. Samples were typically diluted 50 and 100 fold in the assay diluent along with known positive and negative control samples. Mouse anti-human IgG4 antibody (Hybridoma Reagent Laboratory) was diluted 1∶5000 and goat anti-mouse IgG antibody, conjugated to horseradish peroxidase (Southern Biotech), was diluted to 1∶10,000 in the assay diluent. A 3,3,5,5 tetramethyl benzidine, TMB, reagent (Thermo Scientific) was used as the detection substrate and the reaction was stopped using 1N hydrochloric acid after a 15 minute incubation time at room temperature. Results were read using a 96-well plate reader and read at 450 nm. Positivity of the samples was assigned when the absorbance of the sample was above a defined plate-specific cutoff value, the average absorbance of the background (assay diluent only) +3 times the standard deviation of all background wells of the plate (6 per plate).Materials in lateral flow strip: Lohmann diagnostic backing material was used in card format with the dimensions of 300×75 mm. Assay HiFlow Plus (Millipore) nitrocellulose membrane was used in standard 25-mm width. Conjugate pad material was used in a 22-mm width. Absorbent wick was cut to 27 mm strips and buffer pad was cut to 15 mm-wide strips. Compressed cellulose wick material was purchased from Industrial Commercial Supply and cut to 27 mm-wide strips prior to assembly with the other lateral flow strip materials. All materials were assembled with a minimum of 2 mm overlap between materials, in 300 mm × 75–80 mm card format. Lateral flow strips were cut from the cards to a size of 75–80 × 4 mm.An Imagene IsoFlow Reagent Dispenser was used to dispense (0.025 mg) Ov-16 antigen and control antibody  stripes onto the assay membrane and to spray anti-human IgG4 antibody (Hybridoma Reagent Laboratory) conjugated to 40 nm gold colloid onto the conjugate pad material. These materials were assembled on the diagnostics card backing with the absorbent wick. For standard lateral flow strips used either without housing or as comparison tests within housing, a standard cotton fiber absorbent wick material was used; for the assay-actuated lateral flow tests, compressed cellulose wick material was used.The housing top was prepared using a 6-mil acetate film (McMaster Carr) overlay with punched holes. Double-sided tape adhered a 6×8-mm blood separation membrane  to the acetate overlay. Double-sided tape applied to the back of the lateral flow strip backing prior to cutting strips was used to affix the test strips to a larger piece of diagnostic backing material cut to match the size of the housing top. The housing top overlay material with adhered blood filter was then placed on top of the lateral flow strip with the blood filter in contact with the lateral flow strip membrane.Tests run with sera were run directly on the lateral flow strip with no blood cell separation filter. A quantity of 2.5 µl of plasma was pipetted onto the assay membrane and followed by 10 µl of Ov-16 running buffer applied to the same spot. Sample and buffer then were allowed to run into the test for 2 minutes. The test strip was then inserted into a flat-bottom well containing 125 µl of running buffer and the test ran by capillary action. The panel of 449 specimens was run in this manner. For the spiked blood specimen panel, 15-µl blood specimens were applied to the blood filter region of the test and allowed to absorb. The specimen was chased by applying Ov-16 running buffer , 15 µl for Vivid GR filters, and 30 µl for Cytosep 1663 filters, to the blood filter to facilitate sample flow in through the filter. Sample and buffer then were allowed to run into the test for 2 minutes. A total of 125 µl of Ov-16 run buffer was then added to the buffer pad upstream of the conjugate pad, and timing of the test began. Tests were evaluated at 5-minute intervals up to 25 minutes and then in the dry state (24 hours). Clinical specimen tests were monitored at 20 minutes, 1 hour, 24 hours, 10 days, 20 days, 30 days, and 70 days past test beginning.Sera samples were applied to either the conjugate pad upstream of the dried conjugate (BC), directly onto the conjugate (OC), or on the nitrocellulose upstream of the test and control lines (NC). Resulting dry signal strengths were measured using Image J by average pixel intensity across the strip width.2 pieces of materials were cut from the sheets, and pre-exposure thicknesses were measured with microcalipers. The materials were then placed in an environmental chamber and exposed to a programmed temperature and relative humidity for the specified amounts of time at which the thicknesses of the pieces were measured and recorded.Two medical-grade cross-linked polyvinyl alcohol (PVA) sponge sheets  were tested that had different densities and wet thicknesses, either 3 mm or 6 mm. Sheet material of compressed cellulose at 3/8″ was purchased from Industrial Commercial Supply; 1 cmLateral flow tests in housings were prepared as described above, using either Vivid GR or Cytosep 1663 blood filters, paired with either a standard absorbent wick material or compressed cellulose. Both types of wick materials were sized to absorb in excess of the total volume of fluid in the test. Tests were run with varying volumes of blood sample on all types of tests. A minimum of 10 tests, up to 100 tests, were run for each type blood filter, wick material, and volume. Endpoint test failure was decided in cases where blood leakage was present on the nitrocellulose in the region of the test and control line. Since there was a large qualitative range of the amount of blood leakage observed on the membrane, any presence of blood was noted as failure, whether or not the test was readable. Endpoint test failure was determined for the dry state (24 hours) of the test.The sensitivity of most lateral flow assays is strongly influenced by the mixing sequence of the different components of the analyte capture complex. The sequence can be changed by moving the location of the sample input with respect to the conjugate pad and the striped nitrocellulose. When three regions for sample input were tested with sera samples for overall signal development, only the sample input at the nitrocellulose generated specific true positives at the lowest limit of detection (1∶1000 dilution of positive pool), shown in One approach to address the common problem of hemolysis of the red blood cells in the blood separation membrane leaking into the nitrocellulose strip and causing background noise is to break contact between the blood separation membrane and the nitrocellulose after the sera has transferred on to the membrane. A solution was developed in which the blood membrane is detached from the nitrocellulose membrane by the assay fluid and an expanding wick pad without intervention by the test user. The blood separation filter is attached to the top cover of the lateral flow housing instead of being an integral part of the strip , B and DIn the device described, blood filter lift is actuated by expansion of the cellulose at the wicking end of the lateral flow strip. This expansion occurs rapidly when a solution comes into contact with the material and is a desirable feature of the material. However, it could be disadvantageous for the lack of contact to occur before the sample is added to the test, since it would reduce contact and transfer of plasma between the blood filter and the strip when the test is removed from its package. The possibility of humidity in the environment leading to premature expansion was a critical issue that needed to be evaluated before final choice of hydrophilic compressed wick material. The compressed cellulose and two PVA foam materials were evaluated by measuring the height of the material upon exposure to increasing temperature and humidity. All three expanding materials were relatively stable when exposed to moderate temperature and humidity (such as 30°C and 30% relative humidity). However, the two hydrophilic PVA foams (designated by their maximum thicknesses of 3 mm and 6 mm) expanded rapidly (in under 5 minutes) when exposed to high temperature and humidity, similar to those experienced in Africa where onchocerciasis control programs operate at 37°C and 80% relative humidity. In contrast, the compressed cellulose was relatively stable, increasing by only 0.1 mm under the most extreme conditions , despiteThe whole blood test required a separating membrane to transfer plasma to the assay membrane. Two types of blood filters were tested for use in this application. Cytosep 1663 is a thicker, more fibrous material. Vivid GR is a thin, smooth, asymmetric membrane. Both filter blood samples by size exclusion of the red blood cells, allowing the plasma to pass through into the test. However, red cells in contact with surfaces or air will rupture and hemolysis products will no longer be retained by the blood sample filter. For this reason, when the blood filter is placed directly on the optimal location for sample introduction (the nitrocellulose assay membrane), the resulting hemolysis is especially problematic given the proximity to the reading window. Any fluid remaining in contact with these hemolysis products will carry into the lateral flow strip. We examined the failure rate, as defined by presence of red blood cell components onto the membrane, as a function of both the blood filter type, the volume of the blood sample applied, and the type of absorbent wick, either standard or compressed cellulose. Test failures, although varied in both their onset and duration, were observed to emerge as early as 5 minutes into the test and also later than 30 minutes until the test became dry. Only endpoint failure (dry test) was considered for this analysis. A low volume (5 µl) produced no failures, but the blood sample volume is far under the capacity of the filter, resulting in less fluid contact between the filter and the nitrocellulose membrane and a drier sample filter which can draw fluid away from the strip, discouraging transfer of hemolysis products. While lower volume prevents failure, it also results in insufficient transfer of the plasma sample to the nitrocellulose membrane (data not shown), which could negatively impact the test results. The failure rate of tests using lateral flow strips with the standard absorbent wick increased sharply as the volume of blood sample increased, indicating that sample volume had some effect on the failure rate, but there were no observed test failures when the compressed cellulose wick was incorporated into the test strip  with eitA blinded specimen panel of 449 specimens , the tests were also read by two readers at some time points to look at inter-reader variability as well. Three test reads had calls which would result in an equivocal result. These tests fluctuated between very weak positive and negative calls and did not appear to change with respect to time but only with respect to the reader, with different calls made for two of the tests on the same day by two different readers. For test concordance over time and throughout different readers, positive calls of any intensity were considered concordant, and there appeared to be no change in intensity of the positive signal over time. Final test results were called within 20 minutes in 99% of the tests. Only one test was recorded to change from negative result to weak positive between 20 minutes and 1 hour. No tests changed in result from 1 hour to overnight. Shown in Loa loa and lymphatic filariasis specimens, had a sensitivity of 89.1% (95% CI: 86.2%–92.0%) and specificity of 97% (95% CI: 95.4%–98.6%). When compared to an ELISA test designed to detect IgG4 antibodies to the same Ov-16 antigen, the sensitivity and specificity were 96.4% and 93.9%, respectively. Discordance between these two tests only occurred with weak positive specimens where the cutoff of the ELISA and the signal-to-noise ratio in the lateral flow test were the determinants of concordance or discordance. The assay-actuated Ov-16 lateral flow test was evaluated for performance on a panel of 100 blood-spiked specimens, 50 of which were spiked with sera from Ov patients that were microfilaria positive, and 50 of which were spiked with sera from patients that were negative for Ov. The performance of the test was statistically identical to that of the ELISA and the lateral flow test ran with sera samples.A lateral flow test to detect human IgG4 antibodies to the Ov antigen Ov-16 was developed. The Ov-16 lateral flow test, when performed on a panel of 449 specimens including Loa loa-positive sample that resulted in a false positive for Ov-16 was later reported to be from a patient suspected of having onchocerciasis, and one negative was from a field study worker who could also have had exposure. No other Loa loa-positive sample or any of the lymphatic filariasis-positive specimens gave false positives in this sample set. The intended use of the Ov-16 test is to detect exposure to Ov infection in a population that is in post-elimination stage of the disease, primarily in remote areas of Africa. Typically, this will be performed by surveillance teams sampling children under ten years old and in communities that have undergone years of mass drug administration and should no longer be exposed to the parasite. While specificity of the Ov-16 test is high, the use of a test with anything less than 100% specificity in confirmation of elimination will be challenging nonetheless, given that the true positivity rate could approach 0% The sensitivity is similar to that reported for a previously developed  Ov-16 RDT The output for the assay-actuated Ov-16 test is stable to at least 70 days after performing the test, as read by multiple readers. A highly desirable feature for a diagnostics test is a stable test output whereby the test result is the same whether it is read immediately after the test is performed or several weeks after it was performed. If test results are not persistent, a limited read window is required. This limitation results in requiring an additional timing step for the individuals running the tests, which increases the risk for inaccurate reads when taken outside of the window of time, requiring retests in the case of missed windows. Extended test output stability allows programs to assure quality or confirm the data collected during surveillance.A large number of RDTs require limited end-read windows in their product inserts, suggesting instability in the test result. Optimal immunoassay results arise from specifically increasing the relative signal-to-noise ratio. Complex samples such as blood will often contribute to the noise in a test system. Ideally, the analyte can be separated from the sample to reduce the noise in the test result. Lateral flow tests have a small fluid balance with which to perform separation, hydrate reagents, and wash away background and nonspecific interactions. It is common in many gold standard immunoassay techniques, such as dot blots and ELISA, to add sample and detection reagents in a stepwise fashion with large volume washes in between to ensure signal specificity and noise reduction. For the lateral flow test, stepwise and separated sample and buffer additions impact the housing of the test significantly since the sample port must be placed downstream of the conjugate in order for the sample to react with the test line prior to the detection conjugate. In the case of this Ov-16 lateral flow test, the signal-to-noise ratio is optimal if the specimen is allowed to bind to the antigen prior to exposure to the anti-IgG4 gold conjugate. The signal is stable over long periods of time when using plasma or sera only. However, when using whole blood, even with sample filtration, the signal may be obfuscated by eventual hemolysis of the red blood cells in the blood separation membrane and leakage into the assay membrane. This is particularly relevant in this case due to the fact that the specimen needs to be applied upstream to the conjugate pad directly onto the nitrocellulose for optimal sensitivity. The contact of the blood filter with the test membrane limits the read window and test robustness and it also increases dependence on changes in the volume of blood in the filter, the type of filter, and other factors which were not explored in these test runs including damage to the filter, temperature and humidity changes, or buffer volumes outside of the protocol volume. While removal of the blood filter can be achieved by adding a timed step in the test protocol at which the user mechanically removes the blood separation membrane after a short period of time, it is done at the expense of adding significant complexity and risk of user error to the assay procedure. To address this issue with minimal impact on the test procedure as experienced by the end-user, we designed a novel lateral flow test housing in which the blood separation membrane is detached from the nitrocellulose membrane after the plasma has entered the nitrocellulose by the fluids of the assay and an expanding wick material. We demonstrated that the material responsible for the actuation, the compressed cellulose, is robust to typical environmental conditions under which it will be operationally exposed in Africa (37°C and 80% relative humidity).We demonstrated the performance of a user-independent way of detaching the blood separation membrane from the lateral flow test, with the timing consistent with optimal test performance while achieving the highly desirable product feature of an indefinite read window even for weak positives. The elimination of a user step to perform separation is important in reducing user failures. The extended read window is a critical product feature for a diagnostic test that will be used in remote areas in a surveillance context where many people are sampled in a short period of time. The technology is low cost, simple, and robust to high operational temperatures and humidity. The next step is to align this novel approach with manufacturing processes.


Saccharomyces cerevisiae is the first eukaryotic organism for which a multi-compartment genome-scale metabolic model was constructed. Since then a sequence of improved metabolic reconstructions for yeast has been introduced. These metabolic models have been extensively used to elucidate the organizational principles of yeast metabolism and drive yeast strain engineering strategies for targeted overproductions. They have also served as a starting point and a benchmark for the reconstruction of genome-scale metabolic models for other eukaryotic organisms. In spite of the successive improvements in the details of the described metabolic processes, even the recent yeast model  remains significantly less predictive than the latest E. coli model . This is manifested by its significantly lower specificity in predicting the outcome of grow/no grow experiments in comparison to the E. coli model.iMM904 as a basis. We identified and vetted using literature sources 120 distinct model modifications including various regulatory constraints for minimal and YP media. The incorporation of the suggested modifications led to a substantial increase in the fraction of correctly predicted lethal knockouts  from 38.84% (87 out of 224) to 53.57% (120 out of 224) for the minimal medium and from 24.73% (45 out of 182) to 40.11% (73 out of 182) for the YP medium. Synthetic lethality predictions improved from 12.03% (16 out of 133) to 23.31% (31 out of 133) for the minimal medium and from 6.96% (8 out of 115) to 13.04% (15 out of 115) for the YP medium.In this paper we make use of the automated GrowMatch procedure for restoring consistency with single gene deletion experiments in yeast and extend the procedure to make use of synthetic lethality data using the genome-scale model Overall, this study provides a roadmap for the computationally driven correction of multi-compartment genome-scale metabolic models and demonstrates the value of synthetic lethals as curation agents. Saccharomyces cerevisiae is the first eukaryote whose genome was fully sequenced and annotated ) which is identical to the reaction catalyzed by Zwf1p in the cytosol . This suggests that glucose-6-phosphate dehydrogenase activity in the endoplasmic reticulum is not sufficient to compensate for its deficiency in cytosol in an RPE1 mutant background. Interestingly, it has been shown  to render the simultaneous deletion of each of the aforementioned gene pairs/triple combinations lethal  involved in a GPR that implicates genes escribed . In tota PRS1,PRS,PRS4 andin silico essential genes in the minimal medium and nine ESSLs containing five in silico essential genes in the YP medium  and OPI3 (YJR073C) where each gene is essential in vivo [CHO2 catalyzes the first step in conversion of pe_SC (phosphatidylethanolamine) to pc_SC (phosphatidylcholine) in phospholipid biosynthesis pathway (reaction PETOHM_SC in the iMM904 model), whereas, OPI3 catalyzes the last two steps (reactions MFAPS_SC and PMETM_SC). We were not able to fix neither CHO2 nor OPI3 as separate NGGs by any of the methods discussed previously. However, the fact that these two genes form a SL in vivo suggests that they may act as isozymes for reactions PETOHM_SC, MFAPS_SC or PMETM_SC. Interestingly, by mining the literature we found that previous studies have already demonstrated that OPI3 can partially contribute as an isozyme in catalyzing reaction PETOHM_SC [CHO2 OR OPI3). There were no reports implicating CHO2 as an isozyme for catalyzing reactions MFAPS_SC or PMETM_SC. However, previous studies have reported that S. cerevisiae is flexible with respect to phospholipid composition and can substitute pe_SC, ptdmeeta_SC (phosphatidyl-monomethylethanolamine) or ptd2meeta_SC (phosphatidyl-dimethylethanolamine) for pc_SC to a substantial extent [phospholipid compound with the same stoichiometric coefficient as that for pc_SC. Subsequently, we added four hypothetical reactions to the model that produce the phospholipid from any of pc_SC, pe_SC, ptdmeeta_SC or ptd2meeta_SC. Note that these modifications to the model were all global and fixed the inconsistencies for OPI3 as a NGG as well as  as an ESSL mismatch.An interesting example of ESSL mismatches is the gene pair  in vivo . CHO2 caETOHM_SC  implyingl extent -49. In oin silico SL, however, their simultaneous deletion results in a viable strain. SLG mismatches for double gene perturbations can be viewed as NGG mismatches for single gene perturbations in the sense that they both imply that certain functionalities are missing in the model. The only difference between NGGs and SLGs is that the "no-growth" in NGGs is due to the essentiality of single genes whereas in SLGs it is the result of synthetic lethality of gene pairs. Therefore, we simply adapt the same procedure that we used for fixing NGGs to resolve SLG inconsistencies.The SLG inconsistencies represent a mismatch where the two genes form an PGM1 (YKL127W) and PGM2 (YMR105C) that code for isozymes of reaction PGMT (phosphoglucomutase) involved in glycolysis/gluconeogenesis. The simultaneous deletion of PGM1 and PGM2 is lethal based on the iMM904 model since glygogen, a biomass precursor, cannot be produced in the absence of these two genes. Application of the GrowMatch procedure to fix this SLG, did not lead to any literature-supported correction strategy. We next mined the literature for a possible isozyme for this reaction and identified that gene PGM3 (YMR278W) is known to catalyze the interconversion of glucose-1-phophate to glucose-6-phosphate [iMM904 model. The addition of PGM3 to the GPR for reaction PGMT  renders the simultaneous deletion of PGM1 and PGM2 non-lethal.We found and resolved one case of such an inconsistency in both minimal and YP media. This SLG pertains to genes hosphate , howeverin vivo. This implies that deletion of one of these two genes cannot be compensated for by the other gene under the experimental conditions. Therefore, SLES inconsistencies are rectified by suppressing in the model the gene that is not essential in vivo. Essential genes participating in in silico SLs yield GNG inconsistencies. Therefore, the resolution of these GNGs also fixes the SLES mismatches for the double gene mutations. We identified 59 SLES mismatches for the minimal medium and 42 for the YP medium. Fifty one of these SLESs in the minimal and two in the YP medium were fixed by using the global modifications found for GNGs. For example, gene IPP1 (YBR011C) which codes for PPA (inorganic diphosphatase involved in oxidative phosphorylation) forms a SL pair under both minimal and YP medium with gene IPP2 (YMR267W) that codes for the same reaction in mitochondria (reaction PPAm). However, IPP1 has been found to be essential in vivo [IPP2 under aerobic conditions. Interestingly, by investigating the available expression data for these genes we found that the expression level of IPP1 under aerobic conditions with glucose as the carbon source is almost 37 times higher than that for IPP2 [IPP2 is not able to compensate for the deletion of IPP1.The SLES mismatches denote cases where the model predicts that only the simultaneous deletion of both genes is lethal whereas one of the genes (or both) is essential for IPP2 , which mPGK1 (YCR012W), which is associated with reaction PGK (phosphoglycerate kinase) involved in glycolysis/gluconeogenesis, participates in as many as 39 in silico SLs under minimal medium. However, it has been found to be essential in vivo [PGK1 would resolve all these SLESs. Although, we did not find any evidence confirming this resolution hypothesis for any of these genes except for PCK1 (YKR097W), which is known to be suppressed in presence of glucose [PCK1 in the iMM904 model will block production of three biomass precursors, i.e., phe-L , trp-L (L-Tryptophan) and tyr-L (L-Tyrosine) in the absence of PGK1.In another example, gene  in vivo . This im glucose ,53: thisiMM904 model from 12.03% to 23.31%  for the minimal medium and from 6.96% to 13.04%  for the YP medium. It is worth noting that these corrections are based on only the incomplete list of SL data available in literature.Overall, the resolution of model inconsistencies for double gene deletions improved the specificity of in vivo observations, however, the model predictions for supplementation rescue  scenarios are inconsistent with experimental data. We found seven such these inconsistencies under minimal and five under YP medium, respectively, for correctly predicted essential genes, as well as four under both minimal and YP media for SLSL predictions  deletion can grow if the medium is supplemented with folic acid [iMM904 model. By adding folic acid as well as exchange and transport reactions (between cytosol and extracellular environment) to the iMM904 model FOL1 remains essential even though folic acid is allowed to be taken up. GrowMatch suggested addition of any of the two reactions R00937  or R02236 (dihydrofolate:NADP+ oxidoreductase) from the KEGG database to the model so as to connect folic acid to rest of the network. Interestingly, by searching the KEGG database we found that the enzyme catalyzing these two reactions is present in yeast and the gene coding for this enzyme (DFR1: YOR236W) is already present in the iMM904 model.As an example, it has been reported that a strain containing the lic acid . NonetheHMG1 (YML075C) and HMG2 (YLR450W), which forms a SLSL under both minimal and YP media. is an example of auxotrophy mismatches for double gene perturbations: although in vivo observations show that a mutant strain lacking these two genes can be rescued through addition of mev-R  to the growth medium [in silico predictions imply that their double deletion is still lethal even in the presence of mev-R. The addition of an exchange and transport reaction between cytosol and extracellular environment for mev-R to the model resolves this auxotrophic inconsistency. Notably, the addition of this import pathway for mev-R to the model also fixed the auxotrophy inconsistency for the essential gene ERG10 (YPL028W), which is involved in mevalonate biosynthesis.The gene pair h medium , in siliiMM904 model by using all types of inconsistencies for single and double gene perturbations is given in Figure iMM904 model is also available in the Systems Biology Markup Language (SBML) as Additional file Overall, upon including only the global modifications for which a supporting evidence was found, we could fix three auxotrophy inconsistencies for essential genes as well as one for SLs under both minimal and YP media  of the iMM904 model for essentials and synthetic lethals by incorporating a minimum of 112 (out of 120) suggested corrections. All of these modifications are global as they do not invalidate any of the correct model predictions. The proposed corrections span a wide array of changes to the model including relaxation of the irreversibility constraints on existing reactions in the model, adding new reactions, compounds or genes to the model, modifying the biomass equation, changing the GPR associations and medium-specific regulatory constraints. As we found independent corroborating evidence for the proposed corrections, the vast majority  of them remain relevant even for the latest update of the community yeast model (Yeast 4.0) [iMM904 model, twelve were also independently incorporated in Yeast 4.0 whereas five are different from the ones adopted in Yeast 4.0. A comparison of the suggested corrections with Yeast 4.0 is given in Additional file In this study we considered not only essentiality and synthetic lethality predictions but also disagreements in auxotrophy complementation. We also demonstrated that the identified growth-phenotype discrepancies are sometimes due to an incorrect or incomplete ast 4.0) . This inIn addition to model refinement strategies with supporting evidence, we identified more than 60 other global modifications for which there was neither conflicting nor supporting evidence . In addition to all these components the in silico YP medium contains all 20 amino acids and all four nucleotides [his3Δ leu2Δ met15Δ ura3Δ) and supplementation of the growth medium with the missing nutrients at non-limiting but low levels [in silico media [et al [-1h-1 and the aerobic condition was modeled by limiting the oxygen uptake rate to 2 mmol gDW-1h-1 [-1h-1. The flux of non-growth associated ATP maintenance was fixed at 1 mmol gDW-1h-1 [We applied the SL Finder procedure developed by Suthers et al  to the irevisiae  compriseleotides . All simw levels . In addico media . Consista [et al  we chosegDW-1h-1 . The uptgDW-1h-1 . We emplgDW-1h-1  to reconi) relaxation of the irreversibility constraints on reactions in the model, (ii) addition of new reactions from external databases such as KEGG [iii) allowing for direct import/export of metabolites into/out of the cell, and for multi-compartment models [global if they do not conflict with any correct model predictions, and they are called conditional otherwise.The GrowMatch procedure relies on two separate procedures to resolve NGG and GNG growth prediction inconsistencies. The NGGs are fixed one-by-one by minimally perturbing the original metabolic model using three mechanisms including  A single gene k codes for the enzyme catalyzing a reaction j. This can be easily incorporated into our mathematical framework by using the following constraint:(ii) Two genes 1 kand 2 kform a single multi-protein enzyme to enable a reaction j. This case, which is recast as a logic AND relationship between the genes 1 kand 2k, can be enforced by the following set of constraints: The genes 1 kand 2 kprovide isozymes catalyzing a reaction j. This case, indicates a logic OR relationship between the genes 1 kand 2 kto enable the reaction j, and can be mathematically expressed by the following constraints: More than two genes with a combination of AND and OR relationships are required to enable a reaction j. These cases can be mathematically enforced through an appropriate combination of equations (3) and (4). As an example, if three genes 1k, 2 kand 3 kare correlated as (1 kAND 2k) OR (1 kAND 3k) to code for the enzyme catalyzing a reaction j, then the constraints simulating this relationship can be written as following:(The modified GrowMatch formulation to resolve the GNGs (or GSLs) can now be formulated as following:ijs, represents the stoichiometric coefficient of the metabolite i, in reaction j, biomass vdenotes the biomass flux while n, represents the allowable number of knock-outs. This bilevel optimization problem can be solved similarly to GrowMatch and SL Finder [where, L Finder ,25 throuiAF1260 model of E. coli [ΔG [All the model correction strategies provided by GrowMatch serve as hypotheses that need to be tested to confirm their applicability. Different methods were used to test the validity of each type of modification. Similarly to , relaxat E. coli  or metab E. coli  about thcoli [ΔG .-13 as the basis to define high sequence similarity. Finally, the gene suppressions are validated by analysis of the gene expression data as well as searching the literature for available evidence. All the global modifications for which we did not find any supporting evidence using the methods mentioned above, were not incorporated into the model and were just added to the list of modifications with no corroborating evidence.The validity of the added transport reactions to the model are examined by searching the literature to find potential clues about the presence of the suggested transport mechanisms or by querying the databases such as MetaCyc for possibility of presence of those mechanisms in other multi-cellular organisms. The hypotheses for adding new reactions from external databases such as KEGG to the model are tested by performing the bi-directional BLAST between the enzymes catalyzing those reactions and the yeast genome. Consistent with ,20 we asCDM conceived and coordinated the study, participated in its design and helped to draft the manuscript. ARZ performed the simulations and data analysis and drafted the manuscript. Both authors read and approved the final manuscript.The list of model inconsistencies. The complete list of different types of model inconsistencies for single and double gene perturbations along with literature citations.Click here for fileThe list of corrections to the model. The complete list of suggested corrections for mismatches associated with single and double gene perturbations along with supporting evidence. This file also contains details of BLAST analysis for different cases as well as other computations.Click here for fileThe revised yeast model in SBML format.Click here for file