The purposes of this study were to identify distinct latent classes of individuals based on subjective reports of sleep disturbance; to examine differences in demographic, clinical, and symptom characteristics between the latent classes; and to evaluate for variations in pro- and anti-inflammatory cytokine genes between the latent classes. Among 167 oncology outpatients with breast, prostate, lung, or brain cancer and 85 of their FCs, growth mixture modeling (GMM) was used to identify latent classes of individuals based on General Sleep Disturbance Scale (GSDS) obtained prior to, during, and for four months following completion of radiation therapy. Single nucleotide polymorphisms (SNPs) and haplotypes in candidate cytokine genes were interrogated for differences between the two latent classes. Multiple logistic regression was used to assess the effect of phenotypic and genotypic characteristics on GSDS group membership. Two latent classes were identified: lower sleep disturbance (88.5%) and higher sleep disturbance (11.5%). Participants who were younger and had a lower Karnofsky Performance status score were more likely to be in the higher sleep disturbance class. Variation in two cytokine genes  predicted latent class membership. Evidence was found for latent classes with distinct sleep disturbance trajectories. Unique genetic markers in cytokine genes may partially explain the interindividual heterogeneity characterizing these trajectories. Sleep disturbance is a significant problem for oncology patients Newer statistical methods, like latent class analysis and growth mixture modeling (GMM), may allow for the characterization of subgroups of individuals with distinct types of sleep disturbance. These approaches can be used to classify patients with chronic medical conditions like cancer, as well as their FCs who experience the stressors associated with caring for someone with a chronic illness and who have chronic medical conditions themselves. However, only two population-based studies were found that characterized individuals into distinct subgroups based on self-reported sleep disturbance The relationships among sleep, circadian rhythms, and the immune system were the subject of a number of recent reviews Given the paucity of research on the association between cytokine genes and sleep disturbance, the purposes of this study, using GMM in the previously described sample of patients and FCs This descriptive, correlational study is part of a larger, longitudinal study that evaluated multiple symptoms in both patients who underwent primary or adjuvant RT and their FCs Patients were eligible to participate if they: were ≥18 years of age; were scheduled to receive primary or adjuvant RT for one of four cancer diagnoses ; were able to read, write, and understand English; gave written informed consent; and had a Karnofsky Performance Status (KPS) score of ≥60. Patients were excluded if they had: metastatic disease; more than one cancer diagnosis; or a diagnosed sleep disorder. FCs were eligible to participate if they were an adult (≥18 years of age); were able to read, write, and understand English; gave written informed consent; had a KPS score of ≥60; were living with the patient; and did not have a diagnosed sleep disorder.The demographic questionnaire obtained information on age, gender, marital status, education, ethnicity, employment status, and the presence of a number of co-morbid conditions. Medical records were reviewed for disease and treatment information.past month. The global PSQI score is the sum of the seven component scores . Each component score ranges from 0 to 3 and the global PSQI score ranges from 0 to 21. Higher global and component scores indicate more severe complaints and a higher level of sleep disturbance. A global PSQI score of >5 indicates a significant level of sleep disturbance The Pittsburgh Sleep Quality Index (PSQI) consists of 19 items designed to assess the quality of sleep in the past week. Each item was rated on a 0 (never) to 7 (everyday) numeric rating scale (NRS). The GSDS total score is the sum of the seven subscale scores  that can range from 0 (no disturbance) to 147 (extreme sleep disturbance). Each mean subscale score can range from 0 to 7. Higher total and subscale scores indicated higher levels of sleep disturbance. Subscale scores of ≥3 and a GSDS total score of ≥43 indicate a significant level of sleep disturbance The General Sleep Disturbance Scale (GSDS) consists of 21-items designed to assess the quality of sleep in the physical fatigue and energy The Lee Fatigue Scale (LFS) consists of 18 items designed to assess The Center for Epidemiological Studies-Depression scale (CES-D) consists of 20 items selected to represent the major symptoms in the clinical syndrome of depression. Scores can range from 0 to 60, with scores of ≥16 indicating the need for individuals to seek clinical evaluation for major depression. The CES-D has well established concurrent and construct validity The Spielberger State-Trait Anxiety Inventories (STAI-T and STAI-S) consist of 20 items each that are rated from 1 to 4. The scores for each scale are summed and can range from 20 to 80. A higher score indicates greater anxiety. The STAI-T measures an individual’s predisposition to anxiety determined by his/her personality and estimates how a person generally feels. The STAI-S measures an individual’s transitory emotional response to a stressful situation. It evaluates the emotional responses of worry, nervousness, tension, and feelings of apprehension related to how a person feels “right now” in a stressful situation. Cutoff scores of ≥31.8 and ≥32.2 indicate high levels of trait and state anxiety, respectively. The STAI-S and STAI-T inventories have well established criterion and construct validity and internal consistency reliability coefficients attentional fatigue in patients with cancer. Each item is rated on a 0 to 10 NRS. A mean AFI score was calculated, with higher scores indicating greater capacity to direct attention and, therefore, lower levels of attentional fatigue The Attentional Function Index (AFI) consists of 16-items designed to measure Occurrence of pain was evaluated using the Brief Pain Inventory Objective data on sleep-wake activity rhythms were obtained by continuous noninvasive monitoring of activity over 48 hours using a wrist motion sensor  The study was approved by the Committee on Human Research at the University of California, San Francisco and at the second site. Approximately one week prior to the start of RT , patients were invited to participate in the study. If the FC was present, a research nurse explained the study protocol to both the patient and FC, determined eligibility, and obtained written informed consent. FCs who were not present were contacted by phone to determine their interest in participation. These FCs completed the enrollment procedures at home.At the time of the simulation visit, participants completed the self-report questionnaires. Participants completed the symptom questionnaires at 4 weeks after the initiation of RT, at the end of RT, and at 4, 8, 12, and 16 weeks after the completion of RT  In addition, patients’ medical records were reviewed for disease and treatment information.At each of the seven assessments, participants completed the LFS Data were analyzed using SPSS Version 18 Actigraphy files in zero-crossing mode, with 30 second intervals, were analyzed using the Cole-Kripke Algorithm in the Action 4 software (Ambulatory Monitoring Inc) by two of the researchers (KL and CW). First, the file was scanned for missing data. Time limits were set for the 48-hour period. The file was reviewed and intervals were individually set for each day and night period using in order of priority as decision guides: the event marker, diary data, channel data, and cascading movement data.GMM with robust maximum likelihood estimation was used to identify latent classes  with distinct sleep disturbance trajectories  over the 6 months of the study The GMM methods are described in detail elsewhere First, a model with two latent classes was fit to the data, then a model with three latent classes was fit. The process was repeated until the model with a greater number of classes was not supported. Model fit for the GMM was assessed statistically by identifying the model with the lowest Bayesian Information Criterion (BIC), and by testing the “K” versus “K-1″ class models to determine whether a model with K classes fit the data better than a model with K-1 classes with the Vuong-Lo-Mendell-Rubin likelihood ratio test (VLMR) The third index used to evaluate model fit was entropy  IFNG receptor 1 (IFNGR1), IL1R1, IL2, IL8, IL17A, nuclear factor kappa beta (NFKB1), NFKB2, and TNFA. Anti-inflammatory cytokines suppress the activity of pro-inflammatory cytokines and include: IL1R2, IL4, IL10, and IL13. Of note, IFNG1, IL1B, and IL6 possess pro- and anti-inflammatory functions Genomic DNA was extracted from archived buffy coats maintained by the UCSF Genomic Markers of Symptoms Tissue Bank using the PUREGene DNA Isolation System . Of the 287 participants recruited, DNA could be recovered from the archived buffy coats of 253 . No differences were found in any demographic and clinical characteristics between participants who did and did not choose to participate in the study or in those participants for whom DNA could not be recovered from archived specimens.Genotyping was performed blinded to clinical status and positive and negative controls were included. DNA samples were quantitated with a Nanodrop Spectrophotometer (ND-1000) and normalized to a concentration of 50 ng/µL (diluted in 10 mM Tris/1 mM EDTA). Samples were genotyped using the GoldenGate genotyping platform  and processed according to the standard protocol using GenomeStudio . Signal intensity profiles and resulting genotype calls for each SNP were visually inspected by two blinded reviewers. Disagreements were adjudicated by a third reviewer.A combination of tagging SNPs and literature driven SNPs  were selected for analysis. Tagging SNPs were required to be common  in public databases . In order to ensure robust genetic association analyses, quality control filtering of SNPs was performed. SNPs with call rates of <95% or Hardy-Weinberg p-values of <.001 were excluded.As shown in 2) were computed from the participants’ genotypes with Haploview 4.2. LD-based haplotype block definition was based on the D’ confidence interval method Allele and genotype frequencies were determined by gene counting. Hardy-Weinberg equilibrium was assessed by the Chi-square exact test. Measures of linkage disequilibrium , the genetic model that best fit the data, by maximizing the significance of the p-value was selected for each SNP. Logistic regression analysis that controlled for significant covariates, as well as race/ethnicity, was used to evaluate the association between genotype and pain group membership. Only those genetic associations identified as significant from the univariate analyses were evaluated in the multivariate analyses. A backwards stepwise approach was used to create the most parsimonious model. Except for race/ethnicity, only predictors with a p-value of <0.05 were retained in the final model. Genetic model fit and both unadjusted and covariate-adjusted odds ratios were estimated using the STATA software package, version 9. Based on the recommendations of Rothman Ancestry informative markers (AIMs) can be used as a tool to minimize confounding due to population stratification in case-control association studies The majority of the participants were Caucasian (74.7%), well educated (15.9 (±3.0) years), and married/partnered (69.3%). The mean age of the total sample was 61.5 (±11.3) years. The average participant had over four comorbid conditions (4.6 (±2.7) and a mean KPS score of 92.0 (±11.5). Gender was evenly represented within the total sample with 46.2% male and 53.8% female participants. Patients made up 66.4% of the total sample. Approximately 38% of the patients had breast cancer, 49% had prostate cancer, 7% had brain cancer, and 6% had lung cancer. The majority of the FCs (91%) was the patients’ spouses.No significant differences were found between patients and FCs in age (60.9 (±11.6) years versus 62.5 (±10.5) years), KPS score (91.1 (±11.9) versus 93.7 (±10.6)), and number of comorbidities (4.8 (±2.6) versus 4.2 (±2.9)). In addition, at the time of enrollment, no significant differences were found between patients and FCs in their ratings of worst pain (2.0 (±3.2) versus 1.5 (±3.1)), fatigue (4.2 (±2.0) versus 4.5 (±2.0)), sleep disturbance (38.9 (±19.6) versus 38.7 (±16.7)), and depression (9.1 (±8.7) versus 8.3 (±7.2)).Two distinct latent classes of GSDS trajectories were identified using GMM . The fitThe parameter estimates for the two latent classes are listed in No significant differences were found in patients’ (38.9 (±19.6)) and FCs’ (38.7 (±16.7), p = .904) mean GSDS scores at enrollment. However, a significantly higher percentage of participants in the higher sleep disturbance class were patients (100%) than in the lower sleep disturbance class  and precision  of genotype on sleep disturbance class membership , multivariable logistic regression analyses were performed that included the following variables in the models: genotype, age, functional status, and ethnicity ; and three PCs to control for genetic background. Given the fact that FCs were not represented in the higher sleep disturbance class, this variable could not be evaluated in the regression analyses.The only genetic associations that remained significant in the multivariable logistic regression analyses were for IL6 rs35610689  and NFKBIn the regression analysis for NFKB2 rs7898947, after controlling for race/ethnicity, genotype and functional status were the only variables retained in the final model (p = .0066). The overall model explained 12.4% of the variance in GMM latent class membership. Controlling for functional status and race/ethnicity, carrying one or two doses of the minor allele  was associated with a 74% decrease in the odds of belonging to the higher sleep disturbance class (p = .012). Genotype uniquely explained 4.46% of the variance in GMM latent class membership.This study is the first to identify distinct subgroups of oncology patients and FCs based on changes in self-reported sleep disturbance and to evaluate associations between these subgroups and cytokine gene variations. While no differences in total GSDS scores, prior to the initiation of RT, were found between patients and FCs, the higher class was composed only of patients. In addition, these individuals were significantly younger and reported a lower functional status that was not only statistically significant but clinically meaningful  Compared to the lower class, the mean total GSDS score of the higher sleep disturbance class at enrollment represents not only a statistically significant, but a clinically meaningful difference in sleep disturbance score (d = −0.98). Not surprisingly, participants in the higher class reported significantly higher scores on all of the PSQI and GSDS subscales, except for use of sleep medications. While specific studies on sleep medication use in oncology patients and FCs were not identified, previous cross-sectional studies that used either the PSQI or GSDS to evaluate sleep disturbance in oncology patients An interesting finding related to the sleep disturbance phenotype identified in this study was that, except for sleep efficiency and wake after sleep onset, no between class differences were found in any of the objective sleep parameters. On average, participants in both classes slept approximately 6 to 6.5 hours per night. While significantly different, both classes of participants had sleep efficiency scores below the desired 95% that characterizes a good night’s sleep. In fact, an examination of both the subjective scores and objective parameters suggest that both classes of participants had significant amounts of sleep disturbance. For example, participants in the lower class reported problems with sleep quantity and mid-sleep awakenings on more than three days per week. An average of 15 to 16 awakenings per night identified using actigraphy provides additional evidence that all of these participants had problems with sleep maintenance.Consistent with previous reports While initial univariate analyses found between class differences in a number of SNPs for IL6, NFKB1, and NFKB2, after controlling for significant covariates and race/ethnicity, only two SNPs explained a significant amount of the variance in latent class membership. For both NFKB2 (rs7897947) and IL6 (rs35610689), carriers of one or two doses of the minor allele was associated with a decrease in the odds  of belonging to the higher sleep disturbance class. Each SNP explained a significant amount of the variance in GMM latent class membership . Both of these SNPs are located in introns. While the function of these polymorphisms is unknown, they may be surrogates for unmeasured functional polymorphisms that are in linkage disequilibrium with these SNPs.NFKB is a generic name for an evolutionarily conserved transcription factor system that contributes to the effective mounting of an immune response as well as to the regulation of cell proliferation, development, and apoptosis. The NFKB system appears to be activated in stressful situations and in response to tissue damage While no studies were found that demonstrated an association between NFKB and sleep disturbance, three studies have identified associations between IL6 and sleep disturbance It is interesting to note that the SNP in IL6 (rs4719714), which was not significant in this study, was associated with higher levels of sleep disturbance in this same sample of patients and FCs at the time of the patients’ initiation of RT Several study limitations need to be acknowledged. While the sample size for the GMM analysis was adequate Despite these limitations, findings from this study provide preliminary evidence for distinct sleep disturbance phenotypes in oncology patients and their FCs. Because the higher risk phenotype was associated with higher levels of depression and anxiety, as well as higher levels of physical and attentional fatigue, clinicians need to assess for multiple co-occurring symptoms in both oncology patients and their FCs. Finally, the candidate gene associations found in this study suggest a role for inflammation in the development of persistent levels of sleep disturbance.


We studied changes in sediment ecosystem function (i.e. oxygen and nutrient fluxes across the sediment water-interface) by artificially inducing hypoxia of different durations  in a subtidal sandy habitat. Benthic chamber incubations were used for measuring responses in sediment oxygen and nutrient fluxes. Changes in benthic species richness, structure and traits were quantified, while stress-induced behavioral changes were documented by observing bivalve reburial rates. The initial change in faunal behavior was followed by non-linear degradation in benthic parameters , gradually impairing the structural and functional composition of the benthic community. In terms of ecosystem function, the increasing duration of hypoxia altered sediment oxygen consumption and enhanced sediment effluxes of NH4+ and dissolved Si. Although effluxes of PO43− were not altered significantly, changes were observed in sediment PO43− sorption capability. The duration of hypoxia (i.e. number of days of stress) explained a minor part of the changes in ecosystem function. Instead, the benthic community and disturbance-driven changes within the benthos explained a larger proportion of the variability in sediment oxygen- and nutrient fluxes. Our results emphasize that the level of stress to the benthic habitat matters, and that the link between biodiversity and ecosystem function is likely to be affected by a range of factors in complex, natural environments.Disturbance-mediated species loss has prompted research considering how ecosystem functions are changed when biota is impaired. However, there is still limited empirical evidence from natural environments evaluating the direct and indirect (i.e. via biota) effects of disturbance on ecosystem functioning. Oxygen deficiency is a widespread threat to coastal and estuarine communities. While the negative impacts of hypoxia on benthic communities are well known, few studies have assessed  Disturbance to ecosystems has resulted in global declines in biodiversity In addition to the indirect consequences of disturbance for ecosystem functions, which are mediated by changes in the biota, disturbance may also have direct impacts on the ecosystem. For example, physical disturbances such as fire directly affect net ecosystem production (NEP) through emissions, but NEP is also indirectly affected due to reductions in productivity as functional leaf area is lost Marine soft-sediments are among the most common habitats on earth, sustaining highly diverse benthic communities 2 l−12 of the seafloor in coastal zones in situ studies identifying degradation patterns in response to increasing disturbance are scarce Hypoxia  layer. The contribution of the individual species to such processes depends on what functional characteristics it expresses in situ natural benthic community to increasing duration of hypoxia to evaluate its impact on the benthic community and ecosystem functioning. We performed the experiment in an area where benthic structural and functional diversity is naturally low In BEF research, more studies encompassing the complexity of natural ecosystems and thus the realistic consequences of disturbance-mediated species loss are needed Macoma balthica, gastropods belonging to the family Hydrobiidae and the polychaetes Hediste diversicolor and Marenzelleria spp. All necessary permits were obtained for the described field study from Tvärminne Zoological Station.The experiment site was located in the northern Baltic Sea, in the middle archipelago zone of the Hanko peninsula , Finland. The site was 4 m deep and consisted of bare sandy sediments. Salinity in this non-tidal area is about 5.8 and bottom-water temperature was around 17°C during the experiment. The benthic community was dominated by the bivalve 2) to the seafloor. The disturbance simulated patchy hypoxia induced, for example, by drifting algal mats 2S concentrations beneath three independent plastic sheets after 1.5, 3 and 7 days. The thin layer of water beneath the sheets was sampled through  tubes situated in the middle of each sheet.Hypoxia of increasing duration was artificially induced to the benthic habitat by securing black, low-density polyethylene (LDPE) plastic sheets . The replicates were placed in a block design along a 32 m transect, and any plot was separated by at least three meters from the others. Hypoxia was induced for the 48-day treatment in the beginning of June 2008, while the treatments with 3 and 7 days of hypoxia were started in July. The different durations of hypoxia were ended simultaneously, by carefully rolling the plastic away, avoiding disturbing the plots. Sediment color was noted in each plot, and bivalves (shell length >5 mm) at the sediment surface were counted. All manipulations, chamber incubations and subsequent sampling were done using SCUBA.2, height 11.9 cm). To avoid sampling initial sediment reactions, flushing of sediment was allowed for 14 h. This period allowed the sediment to reach a quasi-stable state, but not a complete re-oxidation in situ incubation of ambient water during the experiment. All sampling equipment was acid washed  prior to use.Measurements of sediment oxygen and nutrient fluxes were performed with dark benthic chambers. We chose to exclude any effects of primary production, due to logistical restraints and to control for possible differences in light conditions. After the plastics were rolled away, one chamber frame was pressed 6 cm into the sediment in the centre of each plot, resulting in a final enclosed water volume of ca 6 l (area 504 cm2 and 0.5 ml KI. The rest of each sample was filtered through a Whatman GF/F filter (Ø 25 mm), directly into sample bottles for dissolved Fe (assumed to represent ferroiron (Fe2+), 25 ml) and nutrient analyses (125 ml). All samples were stored on ice during transport to the laboratory. Nutrient samples were frozen (−20°C) until further analysis, and 0.83 ml concentrated HNO3 was added to the Fe2+ samples for preservation before storage at +5°C. Dissolved oxygen concentrations were determined according to the Winkler procedure, while NH4+, NO3− + NO2−, PO43− and dissolved Si (silicate) were measured spectrophotometrically with an autoanalyser (Lachat QuickChem 8000). The results for NO3− + NO2− should be interpreted with caution, as concentrations were near the detection limit . Similarly, the concentration of dissolved iron in the water column was under the detection limit (0.09 µmol l−1) as measured with the ICP-OES  technique. Concentrations of H2S were determined according to Koroleff The water samples were processed on the boat. For determination of dissolved oxygen, 50 ml was fixed with 0.5 ml Mn(OH)43−) sorption properties of the surface sediments were examined for the 0- and 48-day treatments to clarify the effect of hypoxia on the behavior of phosphate in the sediment-water interface. The PO43− sorption measurements , filtrated (0.4 µm Nuclepore PC membranes) and determined immediately for PO43− by spectrophotometer.After incubation, samples for determination of sediment organic matter (OM), total C and N content as well as phosphate sorption properties were taken with cores . The surface sediment (upper 3 cm) was stored at −20°C for later analyses. Sediment OM was determined as loss of ignition (3 h at 500°C). Sediment samples for nutrient analyses were freeze dried (−70°C) and homogenized. Analyses of TOC and TN were performed with a Carlo Erba high temperature combustion elemental analyzer. Phosphate  was determined.After incubation, macrofauna were sampled from each chamber in order to examine their responses to hypoxia and to allow estimation of the possible reduction in their impact on sediment oxygen- and nutrient fluxes. One replicate core  was taken from the central area of each chamber. Finally, all chambers were excavated, in order to account for any deeper-burrowing bivalves. Macrofauna from disturbed sediments were sieved (0.5 mm), sorted and identified alive to species level at 10× magnification. Samples from undisturbed plots were preserved in 70% ethanol and stained with rose bengal for later analysis. To obtain the proportions of juveniles and adults of dominant taxa, shell lengths of bivalves and gastropods, and the width of the 10c = Bi−0.5*Mi*Ri, where Bi = size, Mi = mobility, Ri = reworking mode and position in sediment; modified from Solan et al. Benthic biological traits were used to investigate how the duration of hypoxia affected the functional structure of the benthic community. The selected traits were considered important for sediment nutrient dynamics, and included benthic feeding modes and aspects of benthic bioturbation. This resulted in a total of five traits summing up to 21 different modalities  as plastics were removed. As all bivalves in the 48-day treatment were dead, this treatment was not included. The undisturbed treatment was represented by 20 bivalves extracted from unaffected sediments at the study site. In the laboratory, bivalves from each treatment were put in separate compartments of an aquarium (241×23×30 cm), and their reburial rates (minutes) were measured. The aquarium contained 5 cm sediment and water collected from the experiment site, with temperatures similar to field conditions. Oxygen concentrations were saturated throughout the reburial experiment (≥100%).Behavioral changes were assessed by measuring reburial rates of adult M. balthica collected from the treatments were compared using Kolmogorov-Smirnov 2-sample tests. Sorption isotherms for sediment phosphate were fitted with a power (3 parametric) regression to the plotted data . Differences between curves were indentified with an F-test.One-way analysis of variance (ANOVA) was used to identify differences between treatments for sediment properties, oxygen and nutrient fluxes, if the data fulfilled the requirements of normality (Shapiro-Wilk's test) and homogenous variances (Levine's test). Since no differences in abiotic or biotic parameters could be detected between blocks (p>0.05), block was not included as a factor in the presented analyses. If necessary, data were square root transformed. Any significant differences were further explored with Scheffe's post hoc test and a nonlinear  regression. Size-frequency distributions and reburial rates of Multivariate analyses of benthic community data were performed with the PRIMER software 2 terms), so they were not used. Three models were run: (i) EFM predicted by CPCA alone; (ii) EFM predicted by hypoxia alone; and (iii) EFM predicted by CPCA, after the effect of hypoxia had been removed. The results from the three models were then used to calculate the amount of variability explained by the benthic community and hypoxia alone, and the intersection of effects related to the benthic community and the duration of hypoxia, according to Borcard et al. Finally, the relationship between the benthic community and ecosystem functioning over the stress gradient was tested, with the aim of separating out the responses in ecosystem functioning driven solely by the duration of hypoxia, the benthic community, and the indirect effect of hypoxia mediated by effects on the benthic community. To accomplish this, we used multivariate analyses. Ecosystem functioning was defined as all chemical fluxes, and a similarity matrix based on Euclidean distances of normalized variables constructed. The benthic community was defined as number of species, evenness, number of modalities, average number of species per modality, total abundance and total biomass and a Principal Component Analysis (PCA) was then run on the similarity matrix of Euclidean distances of the normalized data. This analysis had the effect of removing the colinearity that existed between some variables and producing a reduced number of variables to represent the benthic community, i.e., the first three axes of the Community PCA (CPCA) which together explained >90% of the variability in the PCA. Following this, a regression-based multivariate analysis DISTLM, , which i2 l−1) beneath the plastic sheets (the O2 saturation was 0.5±0.1 mg l−1 after 1.5 days and ≤0.1 mg 1−1 after 3 days). After seven days, anoxic conditions (i.e. 0 mg O2 l−1) and formation of H2S (approx. 3 µmol l−1) were observed. Visual observations of sediment color confirmed the decrease in sediment oxygenation with increasing duration of hypoxia. The sediment in the 0- and 3-day treatments was light-colored, while sediments exposed to 7 days of deoxygenation had a partially black sediment surface, probably due to precipitation of ferrosulphides under reducing conditions. An intensely black sediment surface, indicative of complete anoxia, was observed in the 48-day treatment where the sediment also was slightly more compacted than the undisturbed sediments. Sediment content of OM, TC and TN were low, and averaged 0.58±0.05, 0.19±0.02 and 0.02±0.00%, respectively. Sediment OM increased in the 48-day treatment compared to the 0- and 3-day treatments, with 13 and 5%, respectively  had emerged to the sediment surface. Some dead individuals were noted. After 7 days of hypoxia, a larger number of bivalves and polychaetes were observed, and over half of them were dead or flaccid. In the 48-day treatment, only dead bivalves and empty shells remained. Changes in behavior due to increasing duration of hypoxia are illustrated quantitatively by the increasing number of M. balthica observed on the sediment surface  m−2 were found at depth (the figure is based on core sampling and excavation of flux chambers). The increased stress also resulted in decreasing bivalve reburial rates . The bivalves exposed to 7 days of hypoxia had the slowest reburial rate, which differed significantly from the other treatments  in treatments exposed to ≤3 days of hypoxia from the surviving adults in the 7-day treatment . No difference in size was detected between treatments with ≤3 days of hypoxia . The number of species became increasingly variable and slightly declined with increased duration of hypoxia  of the community showed a non-linear, negative response to increasing duration of hypoxia , with the following concentrations of dissolved nutrients; Si: 7.85±0.09, NH4+: 0.39±0.24, NO3− + NO2−: 0.05±0.01, PO43−: 0.18±0.01, Fe2+: 0.07±0.02 µmol l−1. The duration of hypoxia significantly affected sediment oxygen consumption and the efflux of silicate and ammonium. Oxygen consumption was reduced with the duration of hypoxia  was lower in this treatment (5.42 µmol l−1) than in the control , but the observed variability in ecosystem function was also directly related to the duration of hypoxia as well as the benthic community.Healthy communities and a sustained performance of species are essential for retaining the functionality of natural ecosystems. Although disturbance to Earth's ecosystems is increasing, few studies have assessed 2, PO43−) differed between multi- and single species assemblages, and that the functional diversity effects were not a simple summation of individual species effects. However, it is indisputable that other factors than the number of species are of importance when evaluating the impacts of disturbance on the biota and ecosystem functioning. For example, the non-random order of species loss is suggested to affect ecosystem function more severely than extinction patterns expected by chance Numerous studies assessing the consequences of disturbance for ecosystem functioning has focused on the role of declining species diversity. Undoubtedly, the number of species and their identities matter. For example, Waldbusser et al. Macoma balthica exposed to 3 days of hypoxia indicated a sustained performance of this important species. Adult bivalves, which dominated community biomass, are likely to survive shorter periods of hypoxia by closing their valves. A sustained community performance was also supported by the high number of species, abundance, biomass and number of trait modalities still observed in the 3-day treatment. However, the increased variance within these parameters indicated that additional stress might exceed the species' tolerance levels, cf. M. balthica was reduced, community abundance had declined (a reduction of approx. 88% compared to the control), and benthic biomass, although highly variable, began to decrease. Nevertheless, the number of species was not significantly reduced in the 7-day treatment compared to control sediments. Natural disturbances rarely eliminate all individuals in a community, as perturbations seldom are uniform and as spatial refuges might exist sensu Dayton Behavioral changes are initial macrobenthic responses to hypoxia. The benthic fauna in our study were severely stressed after 3 days of hypoxia, as infaunal polychaetes were observed at the sediment surface, and bivalves had extended their siphons and feet. Emergence at the sediment surface may markedly increase the risk of predation, as demonstrated by Norkko & Bonsdorff Although low diversity estuarine communities adapted to stress might have a limited number of functional traits, these might be expressed by several species, enhancing community resilience. Identification of the relationship between functional and species diversity is thus critical in order to predict changes in ecological functions following species loss Macoma balthica, Mya arenaria, Oligochatea, and Marenzelleria spp.; cf. M. balthica and Hydrobiidae. Although such species-identity effects might be crucial for sustaining functioning when facing disturbance The relationship between the performance of species and their vulnerability to a perturbation is essential for understanding the effects of disturbance on ecosystem function Disturbances  often have evident, direct effects on ecosystem functions, but the indirect changes, mediated by alterations in the biota might also be substantial and complex In our study, the duration of hypoxia explained only a minor part (3%) of changes in sediment oxygen- and nutrient fluxes , while a4+ by promoting nitrification, through enlarging the oxic-anoxic transition zone, by transporting solutes into the sediment and by enhancing the rate of microbial processes The benthic fauna is likely to positively affect the release of ammonium from sediments through excretion The low explanatory strength of the duration of hypoxia could partially be due to changes in fluxes during sediment reoxygenation that, in turn, might have contributed to the amount of unexplained variance (49%). For example, the overall degradation of the benthic fauna probably reduced community respiration, which is likely to be the major explanatory factor for the observed decrease in sediment oxygen consumption. However, the increased duration of hypoxia and the co-occurring reduction of the benthic bioturbation potential probably resulted in a reduced RPD-layer Similarly to ammonium, the efflux of silicate increased with the duration of hypoxia, and showed an inverse relation to benthic parameters cf. . High bi3+43− from sediments exposed to hypoxia, probably due to a generally low content of PO43− in this sandy sediment . Multivariate regression-type analyses suggested that changes in ecosystem function were a result of both direct and indirect disturbance effects, of which the disturbance-mediated changes in the benthic fauna explained a major part. Although our system exhibited some resistance towards the increasing duration of hypoxic stress, the gradual degradation in ecosystem functionality suggested that the system might be more vulnerable than it may appear, and that threshold-like shifts in functionality may take place when species tolerance levels are exceeded cf. . Our resIn order to provide relevant information considering the effects of disturbance to conservation and management, we have to translate the results from BEF experiments to predictions on how natural communities respond at appropriate scales Figure S143− additions.Sediment phosphate desorption-sorption behavior after 0 and 48 days of hypoxia at different concentrations of PO Negative values on the y-axis represent desorption from sediment to solution, and the positive values sorption from solution to sediment. Values are given as average ± standard error (N = 4). Isotherms were fitted to replicate values with a power function Click here for additional data file.Text S1Supporting information considering biological trait analysis.(DOC)Click here for additional data file.Text S2 of sediment phosphate desorption-sorption analysis.(DOC)Click here for additional data file.Table S1Biological traits depicting benthic feeding modes and qualities important for community bioturbation. If a species exhibited more than one trait modality, the fuzzy coding procedure was used to assign the species' relative contribution to each modality.(DOC)Click here for additional data file.Table S2 of nonlinear regression analyses and analyses of variance. The effects of increasing hypoxic duration on benthic parameters were illustrated with nonlinear regression analyses Click here for additional data file.


However, relative expression assays depend on the stability of housekeeping gene expression, which can vary depending on certain stimuli. In this study, we analyzed the stability and pairwise variation in the expression of GAPDH, ACTB and RNA18S1 in cultured reproductive tissues under mitogen stimulation. We used fetal membranes, placental villous and umbilical cord explants from patients with normal term pregnancies (>37 weeks of gestation), as well as myometrium and cervix explants from patients undergoing hysterectomies. Tissues were stimulated with lipopolysaccharide or phytohemagglutinin for 24 hours. We then analyzed the expression stability and the pairwise variation of GAPDH, ACTB and RNA18S1 from real time quantitative RT-PCR absolute threshold cycles (Cp) using geNorm software.Intrauterine infection during pregnancy can trigger a local inflammatory response leading to several complications, such as preterm labor. Many studies have used GAPDH and ACTB being optimal in the cervix, fetal membranes and umbilical cord, while GAPDH and RNA18S1 are best for normalization in the placenta and myometrium.In all of the tissues, the three housekeeping genes showed great stability under our experimental conditions. Pairwise variation analyses showed that only two reference genes are required for adequate normalization, GAPDH, ACTB and RNA18S1 are adequate references for gene expression normalization in reproductive tissues stimulated with mitogens in culture.Our results show that  Relative expression analyses, however, require that one or more housekeeping genes be used as references for normalizing the expression of target genes. Some of the most commonly used housekeeping genes are those encoding for glyceraldehyde 3-phosphate dehydrogenase  regardless of the cell type, tissue or the particular experimental conditions.However, several studies have shown that housekeeping gene expression can vary under certain stimuli -7, makinGAPDH was too varied within the placenta and was therefore unreliable as a reference, yet Murthi et al. found it to be stable and suitable enough for normalization [Despite these evidences, the stability of housekeeping genes has been scarcely explored in human reproductive tissues, namely within the placenta and to an even lesser extent within the cervix and myometrium -13. Morelization ,10,11,13GAPDH, ACTB and RNA18S1 within cultured human myometrium, cervix, villous placenta, fetal membranes and umbilical cord after being stimulated with two mitogens, lipopolysaccharide (LPS) and phytohemagglutinin (PHA).In this study, we aimed to analyze the stability and pairwise variation in the expression of This project was approved by the Internal Review Board of the Instituto Nacional de Perinatologia Isidro Espinosa de los Reyes in Mexico City (Registration number 212250–02191) and informed consent was obtained from all participating women.Fetal membranes (n=3), placental villous explants (n=3) and umbilical cords (n=3) were obtained immediately after delivery from the placentae of patients with normal term pregnancies (>37 weeks of gestation), regardless of delivery mode  or the presence of active labor.Myometrium (n=5) and cervix (n=3) samples were also obtained from patients undergoing hysterectomies due to uterine leiomyomas. However, all of the tissue sections used in this study exhibited a normal histology and coloration with no apparent signs of hypertrophy or necrosis.Subsequently, all tissues were transported and then washed in sterile saline to remove blood and debris.3 in measurement, as well as 1 cm2 explants of fetal membranes were cultured for 24 h in DMEM supplemented with 1% lactoalbumin hydrolysate, 1% sodium pyruvate and 1% antibiotic-antimycotic , all reagents from Gibco BRL . Tissues were cultured in 12 well, flat bottom culture plates, with low evaporation lids .Tissue explants of myometrium, cervix, villous placenta and umbilical cord, approximately 1 cmViability of all tissues was confirmed throughout the culture using the Cell Proliferation Kit II (XTT) according to the manufacturer’s protocol .Escherichia coli (055:B5) or 10 μg/ml of PHA from Phaseolus vulgaris . Both stimulated tissues and unstimulated controls were cultured in duplicate.Tissues were stimulated with 1 μg/ml of LPS from After culture, tissues were immediately placed in 1 ml of Trizol  and stored in ice for 30 min to allow the reagent to penetrate the tissue. Samples were then stored at −20°C until further processing.Tissues in Trizol were thoroughly homogenized with sterile scissors and then centrifuged at 12,000 × g for 10 min at 4°C in order to remove insoluble material. The homogenate was recovered and the total RNA was isolated according to the manufacturer’s protocol. Approximately 3 μg of each RNA sample were used to synthesize the complementary DNA (cDNA) with the Transcriptor First Strand cDNA Synthesis Kit , using random hexamer primers. The reverse transcription reaction was carried out in a Mastercycler Gradient equipment  at 25°C – 10 min / 55°C – 30 min / 85°C – 5 min. The synthesized cDNA was stored at −20°C until it was later used.GAPDH , ACTB  and RNA18S1  were designed using the ProbeFinder software accessible at http://www.universalprobelibrary.com. TaqMan probes #60, 11 and 81 were used respectively.Quantitative real-time PCR was performed in a Light Cycler 480 instrument using a Light Cycler 480 Probes Master kit and TaqMan Probes (hydrolysis probes labeled with fluorescein) according to the manufacturer’s protocol . Specific primers for mRNA sequences of GAPDH, ACTB and TNF were designed to have intron spanning sequences, so as to avoid false positive signals from possible residual genomic DNA. Twenty nanograms of sample cDNA were added to each reaction. Real-time PCR conditions were as follows: one cycle at 95°C, 5 min; 55 cycles of denaturation , annealing  and extension .The primers for TNF expression  was also analyzed as an experimental control to ensure that tissues were responsive to mitogen stimulation [TNF relative expression were calculated using the 2-ΔCt method, using each of the three housekeeping genes as references.mulation . Fold chhttp://medgen.ugent.be/genorm/) was used to analyze gene stability and variability, in accordance with the official software instruction. This application calculates the gene expression stability measure for a reference gene (M), as the average pairwise variation for that gene, against all other tested reference genes. Stepwise exclusion of the genes with the highest M value allows for ranking the tested genes according to their expression stability. Genes with the smallest M value (lower than 1.5) are considered the most stable.The geNorm VBA applet for Microsoft Excel (available at Pairwise variation (Vn/n+1) was also calculated in order to evaluate the minimum number of genes required for normalization in comparative gene expression analyses. According to Vandesompele et al., a pairwise variation of 0.15 was considered the cut-off value below which the inclusion of an additional reference gene would not be required [GAPDH, ACTB and RN18S1 of each sample. All samples showed Cp values < 40.Since geNorm requires normalized raw expression levels , we used Light Cycler 480 SW 1.5 software  to obtain absolute threshold cycles (Cp) of Differences in viability of gestational tissues between basal time and 24 h in culture were evaluated with the Wilcoxon test in SPSS v.17 software. Differences with P ≤ 0.05 were considered significant.There was no significant difference in the viability of any tissue throughout the 24 h culture period Figure .TNF relative expression. This behavior was consistent regardless of whether GAPDH, ACTB or RNA18S1 were used for normalization  of housekeeping gene expression after mitogen stimulation in the various reproductive tissues are shown in Table M value of analyzed genes was clearly below the 1.5 threshold, meaning that genetic expression of those genes remained stable independent of experimental conditions.Stability measures  regardless of experimental conditions. Despite the vast number of gene expression studies in human reproductive tissues, expression stability of housekeeping genes has been scarcely explored, especially in the placenta and to an even lesser extent in cervix and myometrium -13.In this study, we aimed to evaluate the expression of some of the most common housekeeping genes in human reproductive tissues  after being cultured and exposed to mitogen stimulation.Mitogens such as LPS are commonly used in experimental models to emulate an intrauterine infection from Gram-negative bacteria and activate the characteristic proinflammatory response that follows. Such a bacterial-induced inflammatory response further induces the expression of several effector molecules, including cytokines, prostaglandins, defensins and matrix metalloproteinases ,16,17.In our experimental setup, reproductive tissues remained metabolically active throughout the 24-hour culture period. Placental and umbilical cord explants tended to decrease their metabolic activity, although not significantly, suggesting that the culture media we used may not be optimal for these particular tissues. In this regard, medium 199 seems to provide more suitable culture conditions for placental explants ,19.TNF relative expression confirmed that all tissues were responsive to the mitogen stimuli.Nevertheless, changes in GAPDH, ACTB and RNA18S1 showed great stability under our experimental conditions in all tissues. In every case, M values were far below the 1.5 stability threshold, proving that these three genes are suitable for the normalization of relative gene expression in human reproductive tissues under mitogen stimulation.Expression analyses of housekeeping genes TBP, SDHA and YWHAZ in normal placental tissues, and from Cleal et al. who found that UBC, TOP1 and YWHAZ were the most suitable housekeeping genes in normal term placentae [Our results differ somewhat from those of Meller et al. who found the most stable genes to be lacentae ,10.GAPDH and ACTB have a high variability in placenta [Our results also differ from a recent application note by LifeTechnologies that shows placenta . HoweverRNA18S1, GAPDH and, to a less extent, ACTB were stable in placentae from intrauterine growth restriction [GAPDH and ACTB in cervical samples [By comparison, our results agree with, Murthi et al. who found that triction . They al samples .GAPDH and ACTB showed a much greater stability than what was reported by the previous authors.However, our experimental approach was different from all these studies since their analyses were performed in freshly obtained tissues while ours were done after tissue culture and stimulation. In these conditions GAPDH and ACTB being optimal in cervix, fetal membranes and umbilical cord, while GAPDH and RNA18S1 are best for normalization in placenta and myometrium.Due to the high stability of housekeeping gene expression, pairwise variation analyses showed that only two reference genes are required for adequate normalization in each experimental condition: GAPDH, ACTB and RNA18S1 are adequate references for gene expression normalization in reproductive tissues which have been stimulated with mitogens in culture.Our results show that The authors declare that they have no competing interests.MAH performed the experiments, analyzed data and wrote the manuscript. RVS designed the study, analyzed data and wrote the manuscript. Both authors read and approved the final manuscript.


This paper discusses sputtered silicon encapsulation as a wafer level packaging approach for isolatable MEMS devices. Devices such as accelerometers, RF switches, inductors, and filters that do not require interaction with the surroundings to function, could thus be fully encapsulated at the wafer level after fabrication. A MEMSTech 50g capacitive accelerometer was used to demonstrate a sputtered encapsulation technique. Encapsulation with a very uniform surface profile was achieved using spin-on glass (SOG) as a sacrificial layer, SU-8 as base layer, RF sputtered silicon as main structural layer, eutectic gold-silicon as seal layer, and liquid crystal polymer (LCP) as outer encapsulant layer. SEM inspection and capacitance test indicated that the movable elements were released after encapsulation. Nanoindentation test confirmed that the encapsulated device is sufficiently robust to withstand a transfer molding process. Thus, an encapsulation technique that is robust, CMOS compatible, and economical has been successfully developed for packaging isolatable MEMS devices at the wafer level. The vast variation in shape and function of MEMS devices make it virtually impossible to create universal packages like those used in integrated circuits, while maintaining the integrity and functionality of the devices. Many application specific MEMS devices such as accelerometers, pressure sensors, and microgyroscopes require custom packaging to function. Microsensors often require contact with the surroundings to measure air or fluid pressure, gas content, or flowing liquids, making device packaging even more challenging and expensive. On top of being geometrically complex to package, moving parts of MEMS devices are highly sensitive to damage and contamination during fabrication and packaging processes. Damage could be caused by chemical contamination, physical touch, or contamination by micro dirt . The cusi)Wafer level packaging: at this level, encapsulation is applied simultaneously on all dies to provide protection during handling, dicing and testing. This type of packaging is usually hermetic.ii)Conventional packaging: after encapsulation is applied, the wafer is diced. Then, each die is picked, placed, and bonded on a leadframe, wire bonded, and plastic molded. This type of packaging is usually non-hermetic.Wafer level packaging is an approach at which the dies are individually encapsulated on wafer level before dicing. Using this approach, MEMS devices generally require two levels of packaging :i)WaferA typical MEMS device consists of sensor or actuator elements fabricated on a silicon substrate. MEMS devices such as RF switches, inductors, filters, and accelerometers do not require interaction with the surroundings to function. Hence, a complete isolation of the sensor or actuator elements would increase device performance as well as its lifetime. A conventional wafer level packaging technique is cap bonding. The bonded cap, however, is commonly thick and occupies a lot of real estate as the cap has to be bonded over and around the movable elements of the device . For thiet al. have pioneered the use of epitaxially deposited polysilicon as an encapsulation structure for piezoresistive accelerometers [et al. studied plasma-enhanced chemical vapor deposition polysilicon germanium as an alternate material for device encapsulation [et al. used permeable polysilicon to fabricate a vacuum shell over movable elements of a MEMS resonator [Various techniques have been developed to encapsulate isolatable MEMS devices. Partridge rometers . In extesulation . Meanwhiesonator . Alternaesonator -7. Howevesonator . The fab2.In order to assess the feasibility of the proposed packaging technique, sputtered silicon encapsulation was built on an unpackaged MEMSTech 50g non-crossing differential capacitive accelerometer . This device is selected due to its size suitability. The device has an adequately large movable elements area for the encapsulation to be built upon. For this reason, viability of the technique on this device would imply its feasibility on other MEMS devices with smaller movable elements area, since all the CMOS compatible processes used could be scaled down. Moreover, the device structure is planar, yet fairly complicated, which made it practical to gauge the potential of this packaging technique.2, which includes bond pad area of 652.3 μm × 2,331.7 μm, and movable elements area of about 500 μm × 700 μm. The encapsulation structure has to cover all movable elements and have a decent base to be built upon. Considering the position of traces and isolation trenches, it was determined that the most feasible size for the encapsulation structure is 1,455 μm × 1,455 μm. Encapsulation of this size would envelop over all movable elements and critical traces while having appreciable base area to stick to. The inner cavity would have a dimension of 1,250 μm × 1,250 μm, and height of approximately 5 μm. The difference between outer and inner dimension, which is 205 μm, would provide a base of width 102.5 μm around the perimeter of the encapsulation structure.The accelerometer device has four identical quadrants of accelerometer fingers, with 84 pairs of beams per side, as shown in C) of a pair of parallel plates is given by:A is the overlapping area of the plates, d is the spacing between the plates, and sε is the static permittivity of the material between the plates. For the 50g accelerometer used, the static permittivity is close to vacuum permittivity (0ε) since the gap between the plates is occupied by air. The accelerometer has an absolute capacitance value of approximately 1 pF.Capacitive accelerometer detects the variance of capacitance resulting from movement of the parallel plates due to impact, as depicted in Tencor surface profiler to check for surface uniformity and existence of microcracks on the structure. After outer encapsulant application, strength of the dome shaped outer encapsulant layer was determined using a Micro Materials'  NanoTest indenter system, which measures hardness and Young's modulus of the outer encapsulant. Seven identical cured encapsulant samples were used in the strength measurement test, with one indent per sample. The final test performed on the encapsulated device was a capacitance test, which was measured using an Agilent 4284A Precision LCR Meter. As shown by Several tests are conducted on the encapsulation structure after fabrication. The encapsulated device was visually inspected for defects and microstructure release using a scanning electron microscope (SEM) and optical microscopy. Then, a profile scan is performed across the encapsulation using a 3.Encapsulation fabrication involves several deposition and etching steps. The process is initialized by sacrificial spin-on glass (SOG) deposition and patterning, and followed by SU-8 base layer formation, sacrificial SOG removal, visual inspection of release etch, silicon main structural layer formation, gold layer deposition, eutectic gold-silicon seal layer formation, and LCP glob top outer encapsulant application, all in the stated order. These process steps are summarized in 3.1.2O) under mild agitation for approximately 24 minutes. Square SOG pattern with narrow rectangles around its perimeter for side etch channels formation was fabricated.Accuglass 314 SOG from Honeywell Inc.  was used as sacrificial layer. This material was chosen due to its ease of application, ease of etching back in hydrofluoric acid (HF), relatively cheap price, good planarization properties, and its similar characteristics to conventionally deposited silicon oxide . Seven c3.2.Immediately after SOG sacrificial layer formation, a single SU-8 (type 2007) layer was spun, patterned, and developed on top of the SOG sacrificial layer. SU-8 is used as the base layer for the encapsulation. The SU-8 layer was spun at 2,000 rpm and consecutively baked at 95°C for 2 minutes. A square mask was aligned over the SOG layer using a UV mask aligner. The mask was aligned such that part of the edges of the SOG perimeter rectangles protruded from underneath the SU-8 layer. The protruding SOG would initiate sacrificial layer removal in the subsequent process step. After post exposure bake (PEB), the SU-8 layer was annealed at 200°C for 3 hours using direct hotplate baking. The annealing would further densify and harden the polymer so that it is adequately strong to endure subsequent furnace annealing step, and function as structural material of the encapsulation.3.3.2O). This solution was found effective in removing sacrificial SOG, while being less damaging to both the SU-8 base layer and silicon structure of the accelerometer [The most critical step in determining the functionality of the device after encapsulation is the release etch. The etch release step was performed using 5% HF solution  sputtering chamber and RF silicon sputtering was performed at 400W forward power with 20 sccm argon flow, at approximately 80°C. The sputtering process was performed for a total of 23 hours and 50 minutes, at pressure level of approximately 14 mTorr, yielding a silicon layer of approximately 25 μm in height. Despite lower deposition rate, this pressure level was selected because of its higher film adhesion due to lower sputtering pressure [Sputtered silicon was used as the main structure for the encapsulation. A silicon mask with square openings was used to pattern the sputtered silicon so as only the area underneath the openings were sputtered. The samples were aligned such that the SU-8 square base structure was at the very center of the mask square opening. The samples were then placed in pressure .3.5.Immediately after silicon sputtering was completed, the samples were transferred into a Baltec DC sputtering chamber for gold sputter deposition. The sputtering process was performed continuously for approximately one hour at a pressure level of approximately 14 mTorr and 40 mA current. Then, the samples were placed in furnace and annealed at 363°C for 3 hours. The annealing served two purposes: (1) to grow a eutectic gold-silicon layer between sputtered gold and sputtered silicon layer, and (2) to anneal the amorphous sputtered silicon into a more densified layer.The gold-silicon eutectic layer serves as the seal layer for the encapsulation. Gold-silicon eutectic layers have been widely used as hermetic bond layers in MEMS -14. Eute3.6.Outer encapsulant needs to be added on top of the encapsulation structure in order to strengthen the encapsulation structure for the subsequent transfer molding process. A thin glob of RTP 3485-1 LCP carbon fiber was melt-dispensed on encapsulated MEMSTech 50g accelerometer using a dispenser heated to approximately 300°C to melt the thermoplastic, yielding an encapsulated device with a uniform glob top after curing. During hardening, slow cooling was used to avoid stress build-up. This thermoplastic was selected as encapsulant material because of its sufficiently high Young's modulus value and its matching coefficient of thermal expansion (CTE) with the outermost gold layer. Gold, which possesses CTE value of 14 ppm/°C, could be excellently matched by the CTE value of LCP carbon fiber, which is on the range of 12-18 ppm/°C . CTE mat4.4.1.Optical microscope picture of encapsulated for MEMSTech 50g accelerometer is shown in 2. Upon closer inspection on an individual completed device, it could be seen that the encapsulation has been squarely fabricated on top of the movable elements of the device  inspection indicates that the accelerometer fingers are completely released upon sacrificial SOG removal using hydrofluoric acid (HF). Moreover, comparable capacitance values before and after encapsulation indicate complete release of the movable fingers. Completed encapsulation structure with LCP outer encapsulant coating is tested for strength using NanoTest indenter system. Nanoindentation test results indicate that the glob top outer encapsulant is sufficiently strong to endure transfer molding process. The encapsulation technique developed in this work has successfully produced robust MEMS packaging using cheap and simple CMOS compatible processes. Solution for some of the common MEMS packaging problems such as process compatibility, custom and complex packaging equipments required, and high cost has been offered for isolatable MEMS devices.This work has presented a novel wafer level packaging technique for isolatable MEMS devices using a simple and economical CMOS compatible microfabrication processes. The encapsulation uses spin-on glass (SOG) as the sacrificial layer, annealed SU-8 polymer as the base layer, RF sputtered silicon as the main structural layer, eutectic gold-silicon alloy as the seal layer, and high strength liquid crystal polymer (LCP) thermoplastic as the outer encapsulant layer. A robust encapsulation is produced only by using and carefully sequencing these CMOS compatible materials and processes, without the need for expensive packaging equipments. The encapsulation could be fabricated on prereleased as well as released MEMS devices. The completed encapsulation consists of a 25 μm thick silicon encapsulation, 8.75 μm thick base layer, 1 μm thick gold layer, and approximately 210 μm thick LCP layer built on movable elements of MEMSTech 50g accelerometer. The layout was 1,900 μm by 1,900 μm square, with a 1,250 μm by 1,250 μm inner cavity area. The encapsulation has 5 μm clearance between the movable elements and the encapsulation structure. Prior to LCP encapsulant application, the silicon encapsulation is tested for surface uniformity using


To compare the effectiveness of three culture media for growth, proliferation, differentiation, and viability of ex vivo cultured limbal epithelial progenitor cells.Limbal epithelial progenitor cell cultures were established from ten human corneal rims and grew on plastic wells in three culture media: supplemental hormonal epithelial medium (SHEM), keratinocyte serum-free medium (KSFM), and Epilife. The performance of culturing limbal epithelial progenitor cells in each medium was evaluated according to the following parameters: growth area of epithelial migration; immunocytochemistry for adenosine 5′-triphosphate-binding cassette member 2 (ABCG2), p63, Ki67, cytokeratin 3 (CK3), and vimentin (VMT) and real-time reverse transcription polymerase chain reaction (RT–PCR) for CK3, ABCG2, and p63, and cell viability using Hoechst staining.Limbal epithelial progenitor cells cultivated in SHEM showed a tendency to faster migration, compared to KSFM and Epilife. Immunocytochemical analysis showed that proliferated cells in the SHEM had lower expression for markers related to progenitor epithelial cells (ABCG2) and putative progenitor cells (p63), and a higher percentage of positive cells for differentiated epithelium (CK3) when compared to KSFM and Epilife. In PCR analysis, ABCG2 expression was statistically higher for Epilife compared to SHEM. Expression of p63 was statistically higher for Epilife compared to SHEM and KSFM. However, CK3 expression was statistically lower for KSFM compared to SHEM.Based on our findings, we concluded that cells cultured in KSFM and Epilife media presented a higher percentage of limbal epithelial progenitor cells, compared to SHEM. Previous studies have shown the maintenance of the corneal epithelial cell mass as determined by a distinct population of unipotent stem cells (SCs), which is probably located in the basal layer of the corneoscleral limbal epithelium ,2. TheseVarious pathologic conditions that affect the ocular surface can partially or completely destroy the limbal epithelial SC repository giving rise to what is called limbal SC deficiency . Total lLimbal epithelial transplantation has allowed us to treat TLSCD with an acceptable anatomic and functional outcome . HoweverAlternatively, ex vivo cultivation and transplantation of limbal epithelial cells have been reported in animal models and in patients with TLSCD with variable results . QuestioIn most cases, the current methods used to establish the cultures do not favor preserving stemness, but promote proliferation and terminal differentiation of TAC ,16,19. TLimbal tissue was obtained from ten human corneal rims of the remaining trephination of in penetrating keratoplasty at the Operating Room at the Department of Ophthalmology, Federal University of São Paulo (UNIFESP). Corneal rims were transported in Optisol GS  to the cell biology laboratory . The research protocol was previously approved by the Institutional Ethics Committee for Research of the Federal University of São Paulo (UNIFESP), São Paulo, Brazil, in accordance with the tenets of the Declaration of Helsinki for experiments involving human tissue.2. The medium was changed three times a week for 4 weeks, and the explants were left in the culture plates during the incubation period.Each donor corneoscleral rim was divided into six equal pieces, in a laminar flow chamber, under aseptic conditions ,20 and uThe following culture media were used to cultivate limbal epithelial cells: A) The first was supplemental hormonal epithelial medium (SHEM), a combination of Dulbecco’s Modified Eagle’s Medium/Ham’s F-12 nutrient mixture  containing 1.05 mM calcium supplemented with 5 µg/ml crystalline bovine insulin , 30 ng/ml cholera-toxin , 2 ng/ml epidermal growth factor , 0.5% dimethyl sulfoxide , 0.5 µg/ml hydrocortisone, 5 ng/ml sodium selenite, and 5 µg/ml apo-transferrin, and supplemented with 10% fetal bovine serum (FBS). All reagents were obtained from Invitrogen Corporation , except those indicated in the text ,21. B) T2 [Cell morphology and the growth area of the limbal epithelial cells were determined in the three culture media conditions (ten different donor explants expanded in each of the three culture media). During the medium exchange, the margin of the epithelium adhered to the bottom of each culture plate was externally marked with a porous pen tip, delimiting the growth cell area. Subsequently, a transparent graph paper was placed under the sheet of the plate to count the previously defined area in mm2 .After 28 days of culture (five different donor explants expanded in each of the three culture media), the epithelial cells were washed three times with PBS  and incubated with Trypsin/EDTA (EDTA) 0.5%  at 37 °C for 5 min, and the detached cells were washed twice with the tested medium containing FBS . Then thAfter being washed five times with PBS, the slides were incubated for 2 h in a moist chamber with the following primary monoclonal antibodies: Ki67  for cell proliferation ,18; cytoAfter 2 h, the slides were washed with PBS, placed in a buffer bath for 5 min, and then incubated with amplification solutions of labeled streptavidin-biotin for 30 min each. The reactions were revealed with diaminobenzidine chromogen solution. The slides were then washed with distilled water, counter-stained with hematoxylin, and dehydrated with 100% alcohol for 15 min each, followed by a bath with xylol for 15 min. Slides were mounted with Entellan  and coverslipped. The proportion of positive cells in each slide was determined by analyzing 200 cells/slide, using a light microscope . All immunocytochemical reagents were obtained from Dako North America, Inc.  [To compare the gene expression of CK3, ABCG2, and p63 among the epithelial cells grown in the culture media, mRNA was extracted and analyzed with real-time PCR techniques after 28 days of culture (ten different donor explants expanded in each of the three culture media). Total RNA was obtained using the TRIzol kit , which lyses cells and dissolves cell components, maintaining RNA integrity. The RNA quality was evaluated with electrophoresis in an agarose gel containing 1% formaldehyde for denaturing conditions.The cDNA was obtained from the total RNA using the reverse transcriptase enzyme. The samples were treated with 1 U/μl of DNase. The reaction was performed in buffer solution with the specific enzyme for the reverse transcriptase enzyme, 0.5 mg/ml polythymine, 0.1 M of dithiothreitol, and 10 mM of each nucleotide. This solution was transferred to a tube containing 2 μg RNA, and finally, 1 μl of the reverse transcriptase enzyme was added. The reaction was performed at 42 °C for 50 min and stopped by raising the temperature to 70 °C for 15 min. The cDNA obtained was then used in PCR to amplify specific fragments of the CK3, p63, and ABCG2 gene.The level of mRNA expression was estimated with quantitative real-time PCR using the GeneAmp 5700 Sequence Detection System SDS , which was developed to detect gene expression with high specificity and sensitivity. The product obtained with real-time PCR was monitored using TaqMan . Relative expression of the gene was calculated using the logarithmic phase of amplification and was correlated to the number of initial copies of gene transcription. Fluorescence for each cycle was quantitatively analyzed with the ABI Prism 7700 SDS (Applied Biosystems). The relative amount of mRNA was estimated using a benchmark calculated from the control group as the average number of cycles determined by the threshold of all samples. The results for the experiments for each group were corrected by the expression of glyceraldehyde-3-phosphate dehydrogenase, which was used as the quantitative endogenous control and expressed in arbitrary units [To evaluate and compare the devitalized throughout cells between the culture media, a quantitative method using Hoechst 33,342 dye (Sigma Aldrich) was applied. The cells were washed twice with PBS, trypsinized, and then neutralized with supplemented medium. The cells were collected in 1.5-ml tubes and centrifuged for 5 min at 1800 rpm, and the supernatant was discarded. An aliquot of 7 μl of the pellet was mixed in 3 μl of Hoechst solution, 10 mg/mL, for 10 min, placed on a slide and coverslipped. The slides were examined with a fluorescence microscope, and positive cells were expressed as a percentage .t test and one-way ANOVA , where non-parametric measure of correlation was performed using the Kruskal–Wallis test with Müller-Dunn’s post-test, were used to compare variables among the three groups. Probabilities of less than 5% were considered statistically significant. For repeated tests, we used the Bonferroni correction to correct for cumulative type I errors.The Student Optical morphological analysis of the limbal epithelial cells cultivated in three culture media is presented in In KSFM, the cells started to grow in 3–5 days, showed faster migration at 19–22 days of culture, and reached confluence in 27.28±4.41 days. The cells grown in this medium displayed epithelial characteristics, a 1:2 nucleus-cytoplasm ratio, but as soon as the migration started, they started to show a 1:3 nucleus-cytoplasm ratio.The cells cultured in Epilife medium grew more slowly; growth began after 3–10 days. Cell growth reached a peak after 19–22 days of culture, and total confluence occurred after 29.22±5.35 days. The cells grown in this medium were more uniform, with a better definition of cell boundaries and a cuboidal appearance with a 1:2 nuclear-cytoplasm ratio.The cell growth area comparison can be better observed in Immunocytochemical analysis  showed tThe results of real-time PCR analysis are presented in Assays for cells devitalized throughout with Hoechst 33,342 showed that cells in the isolated corneal–limbal epithelial sheets remained viable. There was no significant difference in the proportion of devitalized throughout cells among the three culture media  presented in The present study provides evidence that limbal epithelial cells grew differently in the three tested culture media and showed different phenotypes and expression of markers for putative progenitor cells and differentiated cells. In SHEM, growth and confluence occurred earlier, around 24 days of culture when compared to the other groups. As confluence progressed, the cell size and nuclear size increased. Cell proliferation was significantly higher at 12–19 days, but decreased at the end of the experiment to levels no longer significant. This decrease in the proliferation rate was confirmed with the lower percentage of positive cells for Ki67 (43.5%), a cell proliferation marker, in immunocytochemistry. These findings suggest that cultured cell were in the process of higher differentiation.KSFM and Epilife showed significantly slower growth compared to SHEM between 17 and 22 days. Nevertheless, Ki67 gene expression was high at the 28th day of culture. The smaller cell size and slower proliferation observed in KSFM and Epilife are compatible with the less differentiated epithelium reported by Romano et al. , who useABCG2, a member of the ABC transporters, formally known as breast cancer resistance protein 1 (BCRP1), has been identified as a molecular determinant for bone marrow SCs, and has been proposed as a universal SC marker . SimilarHowever, all groups expressed CK3, indicating the presence of differentiated cells, and the KSFM was the medium that less expressed CK3. Although KSFM and Epilife seemed to be more effective in maintaining the limbal epithelial progenitor cells phenotype, the cultures were heterogeneous, and we found CK3-positive cells.In this study, we did not evaluate the colony-forming efficiency in culture media described by Pellegrini  in cultuA notable difference among culture media components is the calcium concentration. KSFM and Epilife, which kept cells in a more undifferentiated state, presented a lower calcium concentration than SHEM.The influence of calcium in cellular differentiation has been known for 30 years and was reported by Boyce and Ham ,31–33. TTransplantation of cultured epithelium that presents more differentiated cells may perceive short-term success due to better cellular adhesion by desmosomes. However, it has the disadvantage of reducing the stemness of the transplanted cells, which can compromise the viability of new epithelia formed in the long term. Sangwan and colleagues  demonstrOthers authors have shown that epithelial cells cultured in low calcium concentration media had a cobblestone-like morphology in a monolayer, with well defined edges, reflecting a lack of calcium needed in the desmosome formation for cell adhesion ,31–33. IAnother important component of culture media is FBS, which stimulates epithelial growth ,35. The Although some of the immunocytochemistry and PCR results were not statistically significant and despite the different trend profiles of cell differentiation in the culture media tested, they all exhibited progenitor cells even after 30 days of culture. Clinical studies comparing epithelial cells in different culture media with various stem cell profiles should still be tested.The use of a 3T3 feeder layer is a precondition for cultivating and expanding limbal epithelial progenitor cells using the cell suspension technique. Varghese et al.  showed tIn summary, cells cultured in KSFM and Epilife presented a higher percentage of limbal epithelial progenitor cells when compared to SHEM. More studies are needed to further characterize the ideal culture condition for limbal epithelial progenitor cells to be used for ex vivo transplantation in ocular surface reconstruction.


To develop a low cost method to screen for virologic failure of antiretroviral therapy (ART) and HIV-1 drug resistance, we performed a retrospective evaluation of a screening assay using serial dilutions of HIV-1 RNA-spiked blood plasma and samples from patients receiving >6 months of first-line ART.pol gene from extracted RNA, sequenced PCR product of positive pools, and used sequences to determine drug resistance. Sensitivity, specificity, and predictive values were determined for different levels of virologic failure based on maximum viral loads of individual samples within a pool.Serial dilution testing was used to assess sensitivity of a simple PCR-based assay . We created blood plasma minipools of five samples, extracted HIV RNA from the pools, PCR amplified the reverse transcriptase (RT) coding region of the HIV-1 Of 295 samples analyzed, 43 (15%) had virologic failure at ≥50 copies/mL . The assay demonstrated 100% sensitivity to detect virus from these four samples, requiring only one round of PCR, and 56% and 89% sensitivity to detect samples with ≥50 and ≥500 copies/mL using two rounds. Amplified PCR products of all positive pools were successfully sequenced and 30% harbored ≥1 major resistance mutation. This method would have cost 10% of the combined costs of individual viral load and resistance testing.We present a novel method that can screen for both virologic failure of first-line ART and drug resistance. The method is much less expensive than current methods, which may offer sustainability in resource-limited settings. Where available, routine HIV viral load testing is recommended to monitor for virologic failure of antiretroviral therapy (ART) The objective of virologic monitoring is to determine if a patient with viremia has failed ART due to drug-resistant virus or if first-line therapy may be salvaged (e.g. by increasing adherence or eliminating interacting medications). While drug resistance is more likely with higher levels of viral replication and low levels of viremia are often due to “blips” pol gene, and sequencing of the PCR product for detection of drug resistance mutations. Use of pooled rather than individual samples decreased assay sensitivity to a level at which virologic failure was likely  and drug resistance could be assessed. We hypothesized that this method would be useful to screen for both virologic failure of first-line ART and drug resistance in most resource-limited settings.In order to lower the costs associated with identifying virologic failure and drug resistance in the setting of ART, we designed a platform combining sample pooling with qualitative polymerase chain reaction (PCR) amplification of the reverse transcriptase (RT) coding region of the HIV-1 This study was approved by the UCSD Human Research Protections Program. All participants whose samples were used for this study signed a written informed consent.Testing was performed on pooled patient samples and serial dilutions of HIV RNA-spiked HIV-negative blood plasma. For serial dilution testing, a standard solution obtained from the NIH AIDS Reagent Program with a viral load of 150,000 HIV RNA copies/mL was used. Serial dilutions were prepared by combining this solution with HIV-negative blood plasma to obtain samples with HIV RNA copy numbers of 50, 100, 500, 1,000, and 5,000 copies/mL. These serial dilutions were made in triplicate and designed to simulate minipools (consisting of five samples) containing a single specimen of 250, 500, 2,500, 5,000, and 25,000 copies/mL (and four samples with zero copies/mL).Blood plasma samples collected from participants in the San Diego Primary HIV Infection Program enrolled between January 1998 and January 2007 were used. These participants had initiated ART with at least three agents for ≥6 months (+/−2 weeks). Each sample had previously undergone individual quantitative viral load testing using the ultra-sensitive Amplicor HIV-1 Monitor viral load assay . Technicians performing the assay were blinded to patients and viral load results. Multiple samples from some patients were included because of planned 6-month follow-up visits, but each of these samples was obtained at a different time point.Starting volumes of 500 microliters were used for the serial dilutions and the sample pools that were constructed by combining 100 microliters of 5 individual patient samples. Testing on minipools of 5 patient samples rather than individual samples was performed to decrease assay sensitivity to detect individual patient samples containing ≥1,000 HIV RNA copies/mL, in addition to the expected cost savings from pooled testing. A pool size of 5 was chosen, since it is less technically complex than larger pool sizes and allows for a sufficient volume of each individual sample to be included in the pool. RNA extraction was performed using the QIAmp Viral RNA Purification Spin Protocol  with a final elution volume of 50 microliters. Reverse transcription was performed using the Ambion® RETROscript kit , according to the manufacturer's instructions.S3).To develop the qualitative pooled RT assay, the PCR protocol used the following hemi-nested primers in the HIV-1 RT coding region, which are specific to regions that are conserved across clades (See First round:GGAAGAAATCTGTTGACTCAGATTGG (Forward)CI-POL1 ACCCATCCAAAGGAATGGAGGTTCTTTC (Reverse)3RT Second round:AAATCCATACAATACTCCAGTATTTGC (Forward)5RT ACCCATCCAAAGGAATGGAGGTTCTTTC (Reverse)3RT For the first round of PCR, 10 µL of cDNA product were added to 40 µL of reaction mixture that consisted of 0.5 µL of Taq polymerase (Roche), 5 µL of 10× PCR buffer with magnesium chloride (Roche), 1 µL of 10 mM dNTPs (Roche), 31.5 µL of nuclease-free water, and 1 µL each of the CI-Pol1 and 3RT primers. The 50 µL solution was then heated to 95°C for 2 minutes, followed by 35 cycles at 95°C for 30 seconds, 50°C for 30 seconds, and 72°C for 1 minute and a final heating step at 72°C for 10 minutes. For the second round PCR, 5 µL of the first round PCR product was added to 45 µL of reaction mixture that consisted of the same reagents and cycling conditions as for the first round of PCR, except for a slightly larger volume of nuclease-free water (36.5 µL), and 1 µL each of the 5RT and 3RT primers. One round of PCR was performed on the serial dilutions of RNA in spiked HIV-negative blood plasma, and both rounds of PCR were performed on the minipools. The presence or absence of PCR product was assessed using agarose gel electrophoresis after each round. Although this platform is designed for deconvolution of positive pools by retesting of individual samples included in pools with detectable PCR product, this was not performed for this proof-of-principle experiment due to limited stored sample volumes. The amplified PCR product for each positive pool was then sequenced using a “home brew” Sanger sequencing technique, as described elsewhere For serial dilution testing, amplifications were performed in triplicate and repeated, and detectable PCR product was evaluated at each input viral load level. For the patient samples, we determined the sensitivity, specificity, and positive and negative predictive values for the first- and second-round PCR reactions to detect varying levels of viremia  constituting the minipools based on previous viral load testing of individual samples. Cost savings were estimated based on the efficiency of the assay at each threshold and the cost of viral load assays and drug resistance testing at University of California, San Diego.For the serial dilutions of RNA in spiked HIV-negative plasma, first-round PCR product was detected for all replicates with viral loads of 5,000 and 1,000 HIV RNA copies/mL. For patient samples, 295 blood plasma samples from 171 patients were included for 295 unique patient visits. The majority (95%) of the cohort was male, with a mean age of 37 years (range 21–62 years), and most (90%) were men who have sex with men see Table 1. and The qualitative pooled RT assay was performed on 59 minipools of 5 patient samples (N = 295 samples). Of these, 39 minipools yielded no PCR product after two rounds, 3 yielded product after the first round, and 17 yielded product only after both rounds. The assay detected individual samples with ≥1,000 HIV RNA copies/mL with 100% sensitivity using one or two rounds of PCR . The sensitivities of the first- and second-round PCR were 9% and 56% to detect samples with ≥50 copies/mL and 33% and 89% to detect samples harboring ≥500 copies/mL. Negative predictive values were around 90% or higher for both rounds of PCR to detect ≥500 copies/mL. The test characteristics of the pooled RT assay to detect varying levels of virologic failure in individual samples using one and two rounds of PCR are displayed in Sequence data for the HIV-1 RT coding region were successfully generated from each of the 20 positive minipools. Of these sequences (pools), six (30%) had mutations associated with low-, intermediate-, and/or high-level resistance to nucleoside or non-nucleoside reverse transcriptase inhibitors. The most common mutation was K103N, followed by mutations at positions 184  and 215  of HIV-1 RT. The cost of sequencing amplified PCR product, including PCR clean-up, was $87 per sample, compared to around $300 for commercial genotyping. Therefore, the total cost of sequencing the PCR product of positive pools using the pooled RT assay would have been $1,739, compared to $6,000 for commercial genotyping.pol gene using primers that are conserved across clades, since the vast majority of first-line ART regimens in resource-limited settings are comprised of NNRTI and NRTIs. This allows for the PCR product to be sequenced for the presence of the mutations most likely to be associated with ART failure in these settings. This platform, however, can also be adapted to evaluate populations using protease inhibitor (PI) based therapy by adding an additional PCR assay using primer sets that cover the HIV-1 pro coding region.Although quantitative viral load testing on pooled specimens has shown promise in reducing the cost of virologic monitoring of ART in resource-limited settings Unlike quantitative pooling methods Further, our platform detected all samples with ≥1,000 HIV RNA copies/mL with negative predictive values of 100%, even when only one round of PCR was performed, and requiring only one round of PCR would greatly decrease the complexity and cost of the assay. One round of PCR also offered an advantage over two rounds with respect to positive predictive value, although three pools with drug resistance mutations were only identified after two (not after one) round of PCR. Due to the qualitative nature of the assay, it was not possible to differentiate between samples that had lower and higher viral loads. At higher threshold levels for virologic failure, the false positive rate increased (for round two) due to the detection of samples with detectable viral loads that were below the set threshold for virologic failure. Therefore, the positive predictive value of the assay decreased as the threshold for virologic failure was raised when both rounds of PCR were performed. Since two rounds of PCR did not offer an advantage over one round for the detection of ≥1,000 HIV RNA copies/mL and had a higher false positive rate, we hypothesize that optimization of both assay sensitivity and positive predictive value will be achieved more easily using one round of PCR, although this should be validated in larger clinical cohorts. On one hand, the use of pools contributes to a higher false positive rate, but on the other hand it contributes to higher efficiency and lower costs. The impact of each should be investigated further in settings with a spectrum of different prevalence of virologic failure.Concerning the detection of HIV-1 drug resistance, the PCR products of all of the positive minipools were successfully sequenced, and approximately one third of these harbored one or more mutations conferring some degree of resistance to reverse transcriptase inhibitors in one or both classes. When used in real time, sequencing would be performed on the PCR product of individual samples after deconvolution in order to give an individualized account of virologic failure and drug resistance profiles. Of course, these methods require laboratory settings with technical expertise in PCR and sequencing, which already exist in many resource-limited settings but are not used outside of the research setting due to cost. This combined lower-cost method would, therefore, allow for virologic monitoring and drug resistance genotyping to be made available for routine patient care in many of these areas and allow more informed decisions regarding when to change ART.A major limitation of this study was a low prevalence of virologic failure among the study patients, of whom only four had viral loads above 1,000 copies/mL. Since the objective of this platform is to screen for levels of viremia likely to represent true ART failure , further evaluation using more samples with viral loads within the range of interest is needed to define the true sensitivity of the assay. Although the presented data indicate that the sensitivity of this assay is close to the desired target of at least one individual sample in the pool with 1,000 HIV RNA copies/mL using only one round of PCR, this protocol may require further optimization. For example, if the first-round PCR does not prove effective in detecting pools with at least one sample with ≥1,000 copies/mL in future applications, both rounds of PCR may be used to screen the minipools. Alternatively, if the assay is too sensitive, additional dilution with phosphate-buffered saline or another agent may be performed after pooling to achieve the desired sensitivity. Pool size may also be increased to lower sensitivity and cost, or a combination of increased pool size and further dilutions may be employed. The utility of these changes would mostly depend upon local factors, such as the prevalence of virologic failure in the population, the definition of virologic failure used by national ART committees and policymakers, and the number and frequency of samples screened. In addition, this study used banked specimens with different storage times, and there is the potential for RNA degradation in some samples that may have affected the results, and screening in real-time would be helpful to determine if this assay has the desired sensitivity or requires further optimization. Finally, since this study was performed using samples from a U.S. cohort, no patients infected with non-B subtypes were included in the analysis. Although the PCR primers used in this platform span a region in the RT coding region that is conserved across clades, future experiments should focus on the predominant infecting subtypes in the resource-limited settings that may benefit from the proposed methods.In conclusion, we present a platform that incorporates sample pooling, PCR of the HIV-1 RT coding region and sequencing of generated PCR product that can be used to screen for both virologic failure of ART and drug resistance. Although further optimization of assay sensitivity and evaluation on non-B HIV-1 subtypes are needed, the lower assay costs and ability to combine virologic monitoring with drug resistance testing make it an attractive alternative to commercial assays. If proven effective, these methods would allow for maximization of first-line ART and decrease complications associated with delayed detection of ART failure in areas with limited resources for HIV services.Figure S1CI_POL1 alignment with reference sequences for HIV-1 subtypes .(DOCX)Click here for additional data file.Figure S23RT alignment with reference sequences for HIV-1 subtypes .(DOCX)Click here for additional data file.Figure S35RT alignment with reference sequences for HIV-1 subtypes .(DOCX)Click here for additional data file.