High rates of recurrent tuberculosis after successful treatment have been reported from different high burden settings in Sub-Saharan Africa. However, little is known about the rate of smear-positive tuberculosis after treatment default. In particular, it is not known whether or not treatment defaulters continue to be or become again smear-positive and thus pose a potential for transmission of infection to others.To investigate, in a high tuberculosis burden setting, the rate of re-treatment for smear-positive tuberculosis among cases defaulting from standardized treatment compared to successfully treated cases.Retrospective cohort study among smear-positive tuberculosis cases treated between 1996 and 2008 in two urban communities in Cape Town, South Africa. Episodes of re-treatment for smear-positive tuberculosis were ascertained via probabilistic record linkage. Survival analysis and Poisson regression were used to compare the rate of smear-positive tuberculosis after treatment default to that after successful treatment.A total of 2,136 smear-positive tuberculosis cases were included in the study. After treatment default, the rate of re-treatment for smear-positive tuberculosis was 6.86  per 100 person-years compared to 2.09 (95% CI: 1.81–2.41) after cure . Among defaulters, the rate was inversely associated with treatment duration and sputum conversion prior to defaulting. Smear grade at start of the index treatment episode  was independently associated with smear-positive tuberculosis re-treatment, regardless of treatment outcome.In this high-burden setting, there is a high rate of subsequent smear-positive tuberculosis after treatment default. Treatment defaulters are therefore likely to contribute to the pool of infectious source cases in the community. Our findings underscore the importance of preventing treatment default, as a means of successful tuberculosis control in high-burden settings. A major principle in tuberculosis control is the necessity to ensure that patients adhere to a full course of treatment. At least six months anti-tuberculosis multidrug chemotherapy is required to achieve cure in smear-positive tuberculosis patients with initially drug-susceptible disease Adherence to a full course of treatment is therefore expected not only to prevent disease recurrence, but also to contribute to a reduction in tuberculosis burden at population level Non-adherence to a full course of anti-tuberculosis treatment is usually termed ‘treatment default’, defined as interruption of treatment for at least two consecutive months. Risk factors for treatment default such as lack of knowledge and family support, distance between home and health care facility, and drug side effects have been widely studied While treatment defaulters could be specifically targeted by interventions to prevent default, to retrieve those who have defaulted and to prevent subsequent recurrence of disease This study was conducted in a setting with a high tuberculosis burden in South Africa. High rates of recurrent tuberculosis after successful treatment have been reported from this and other settings in Sub-Saharan Africa. Exogenous re-infection rather than relapse seems to be the major underlying cause of recurrence in successfully treated cases In the context of frequent tuberculosis re-infection, little is known about the significance of treatment default as a risk factor for smear-positive tuberculosis.The objective of this study was to investigate the rate of re-treatment for smear-positive tuberculosis, after defaulting from an initial treatment episode. We hypothesised that in a setting with a high tuberculosis burden, sputum smear-positive tuberculosis cases who default from treatment are more likely to return for treatment with smear-positive disease compared to those who successfully complete their treatment. We further aimed to investigate whether treatment duration and sputum conversion prior to default are associated with re-treatment for smear-positive tuberculosis.2 with 36,000 inhabitants of low socio-economic status and a high-burden of tuberculosis in metropolitan Cape Town, South Africa Two adjacent urban communities covering an area of 3.4 kmTreatment outcomes were documented by local health care staff according to standard definitions Routine program data entered by local health care staff from both clinics into paper-based tuberculosis treatment registers for the years 1996 to 2008 were captured in an electronic database. Missing values for treatment end date and treatment outcome were ascertained as follows: If no end date (default date) was documented in the treatment register, the patient file was reviewed and the last day at which the patient was documented to have taken up medication was accepted as the end date. If no treatment outcome was documented in the register, patient files were reviewed and the documented treatment outcome was updated in the database.st October 2008, in order to allow for sufficient time to re-treatment (see below).A retrospective cohort study was conducted, comparing sputum smear-positive tuberculosis cases who successfully completed treatment to those who defaulted from treatment between 1996 and 2008. From the database, we selected all episodes of treatment with a documented smear-positive sputum result at start of treatment and a treatment outcome of either cure, treatment completed or treatment default. ‘Sputum smear-positive’ was defined by at least one sputum sample documented smear-positive for acid-fast bacilli by fluorescence microscopy. Episodes of second-line treatment, episodes of treatment for smear-negative tuberculosis, episodes with treatment outcomes other than success or default and those without a treatment outcome documented, were not included in the study. We further excluded episodes with a treatment end date after 31All treatment episodes included in the study were defined as index episodes. An episode of re-treatment for smear-positive tuberculosis (study outcome) was defined as any subsequent episode of treatment for documented smear-positive tuberculosis recorded for the same individual person, with a minimum time of two months between the end date of the index episode and the record date of the re-treatment episode.We used Registry Plus™/Link-Plus Matches of treatment episodes identified from the record linkage were initially reviewed and classified on the basis of concordance of the matching variables into perfect matches, highly probable matches and probable matches: A ‘perfect match’ was defined by 100% concordance of each, first and surname, sex and year of birth. ‘Highly probable matches’ had minor spelling differences in first- or surname and 100% concordance of both, sex and year of birth. ‘Probable matches’ had 100% concordance or minor spelling differences of first- and surname and 100% concordance of either sex or year of birth but discordance or a missing value of the remainder of the two. Matches identified by the software that did not meet these criteria were discarded.In order to ensure that index and re-treatment episode belonged to the same individual person, all perfect, highly-probable and probable matches underwent careful manual review using the original paper-based treatment registers and, if necessary, patient folders as a reference. In line with the study definition for episodes of re-treatment for smear-positive tuberculosis, matches were excluded if the time period between the end date of the index episode and the record date of the re-treatment episode was shorter than two months.STATA™ 10.1 statistical application  was used for data analysis. Survival analysis and Poisson regression were used to determine rates of re-treatment for smear-positive tuberculosis after treatment default vs. after cure/completion. Cases entered the study on the date when they successfully completed or defaulted from the index treatment episode and were censored on the date when the first re-treatment episode (smear-positive) was recorded in the registers or else at the end of the study period. A multivariable regression model was developed using a step-wise forward technique: co-factors documented at the index episode were considered if they reached P<0.10 significance at univariable regression and resulted in a change of +/−0.1 Rate Ratio for the principal determinant under study. Among treatment defaulters, sub-group analysis was conducted, taking into consideration treatment duration, and sputum conversion prior to defaulting, as documented at the end of the intensive phase of treatment (month 2–3).Permission to access the program data for research has been granted by the City of Cape Town (research ID = 10142), the custodian of the data. The study was approved by the Committee for Human Research, Faculty of Health Sciences, Stellenbosch University (N09/05/144 and amendments 1 and 3) and also by the Ethics Advisory Group of the International Union Against Tuberculosis and Lung Disease. This was a retrospective analysis of routine data and therefore we requested and were granted a waiver of individual informed consent from the ethics committee. The use of a unique subject ID ensured anonymous analysis. Only the senior data staff had access to personal identifiers which were removed after the record linkage process.A total of 2,521 tuberculosis cases with an episode of first-line treatment for smear-positive tuberculosis were recorded in the treatment registers 1996–2008. Of these, 2,136 were included in the study, 1,743 with documented cure, 126 with treatment completion and 267 with treatment default. A breakdown of cases included and excluded is shown in Of all cases included, 1,274 (59.6%) were male, median age was 34 years, and 1,065 (49.9%) had an HIV test result documented, 110 (10.3%) of whom were HIV positive.For 291 (13.6%) of the 2,136 smear-positive cases included in the study, an episode of re-treatment for smear-positive tuberculosis was identified via record linkage and confirmed via manual review. A detailed overview of the record linkage and manual review is shown in Median time between the end date of the index episode and the record date of the subsequent episode was 17 months. Ninety-two of the 291 cases with re-treatment for smear-positive tuberculosis (31.6%) had defaulted at the index treatment episode.The rate of re-treatment for smear-positive tuberculosis was 2.09  per 100 person-years after cure, 2.19 (95%CI: 1.29–3.69) after treatment completion, and 6.86 (95%CI: 5.59–8.41) after previous treatment default.For tuberculosis cases defaulting during the index episode, the unadjusted Hazard Ratio (HR) of re-treatment was 3.29 (95%CI: 2.56–4.22) using cases with cure as the baseline . Sputum In cases after treatment default, there was a steep increase in the cumulative rate of smear-positive tuberculosis within the first two years. By the end of the second year, 27.9% (95% CI 22.8% –33.8%) had experienced a re-treatment episode, compared to 5.8% (95%CI: 4.7%–7.0%) of cases after cure. The failure function for cases after default differed according to smear-grading at start of treatment  .Among the 267 treatment defaulters, 17 (6.4%) defaulted during the intensive phase of treatment. There was an inverse linear association between the time to treatment default (i.e. months of treatment prior to defaulting) and the rate of re-treatment .Of the treatment defaulters, the sputum smear result at the end of the intensive phase of treatment was negative in 149 (55.8%), positive in 43 (16.1%) and not documented in 75 (28.1%). The rate of smear-positive tuberculosis re-treatment was 5.81 (95% CI: 4.34–7.78) per 100 person-years among defaulters with documented sputum conversion prior to defaulting, 6.07 (95% CI: 3.66–10.07) with documented positive sputum smear result at follow-up, and 10.00 (95% CI: 7.07–14.14) among treatment defaulters without documented follow-up sputum smear-result. Thirty-four (37.0%) of the 92 re-treatment tuberculosis cases who defaulted from treatment at the index episode, defaulted again from re-treatment, compared to 17 (8.5%) of 199 re-treatment cases defaulting after previous cure or treatment completion .This study demonstrates for the first time the substantially high rate of smear-positive tuberculosis associated with previous treatment default. Treatment default remains a major risk factor for subsequent smear-positive tuberculosis, even in settings where tuberculosis recurrence due to re-infection is generally common.The rates of smear-positive tuberculosis presented here are based on treatment records and likely underestimate the true rates of smear-positive tuberculosis after cure, treatment completion and default, because some of the tuberculosis cases might not have returned for treatment or they might have sought treatment elsewhere, and because the population at risk in this study includes those who might have moved or died.The differences in the rates of smear-positive tuberculosis after treatment default vs. after success are due to the strikingly higher rates within the first two years in those defaulting from treatment. Our results suggest that tuberculosis had been initially contained in the majority of treatment defaulters, i.e. those with longer time to default and with documented smear conversion, and worsened very soon after stopping the treatment. Cases defaulting earlier from treatment and those who continue smear-positive most likely continue to suffer from active disease resulting in high rates of re-treatment within the first two years after defaulting. Absence of smear examination recorded at the end of the initial phase may be a marker of irregular clinic attendance prior to default.Treatment default is widely considered a risk factor for disease re-activation, but very few studies have looked at rates of tuberculosis after treatment default. A study on re-infection among successfully treated cases in the same communities 1993–1998 documented the frequency of culture confirmed tuberculosis in a sample of previous treatment defaulters for whom strain type information was available, similar to those we found The rate of recurrent smear-positive tuberculosis after treatment success corresponds well with previous findings from the Western Cape Province The more constant rate of recurrent episodes after treatment success suggests that recurrence in this group may be more likely due to re-infection, as suggested previously The extent to which smear-positive cases after both, previous default and previous success, maintain the tuberculosis epidemic in this high-burden community cannot be determined from the results of this study. Den Boon et al. previously showed in a prevalence survey conducted in the same setting, that 56% of previously undetected prevalent smear-positive tuberculosis cases had a history of previous treatment Although the rate of smear-positive tuberculosis among treatment defaulters is higher, cases after treatment success account for the vast majority of recurrent smear-positive tuberculosis in this setting, a finding that is similar to those reported by Wood et al. in a study using notification data from Cape Town We did not find evidence in our study that the rate of smear-positive recurrent tuberculosis was higher among HIV co-infected cases. HIV infected individuals may be less likely to be smear-positive upon recurrence and those living with HIV might be more likely to die, reducing the ‘person-years at risk’ which would result in lower rates.Sputum smear grade at start of treatment was associated with smear-positive recurrence, independent of the later treatment outcome – similar to findings reported by Hesseling et al. in a cohort study from the same community using 24 months follow-up Our study has limitations. We made use of probability record linkage in order to identify subsequent episodes of treatment among individuals. Although this method is considered sensitive Residual confounding might have occurred in our study, given that we were unable to control for other factors known to affect the risk of tuberculosis recurrence, such as smoking, persistent cavities, or undetected drug-resistant disease Further, this study is an evaluation of routine health services based on the information that existed within those services. Such information might be less accurate than information prospectively collected as part of a research project. As noted above, the study did not take into account patients who, for whatever reason, did not return to the health service.We show for the first time that in a high-burden setting, tuberculosis cases defaulting from their treatment are at high risk of subsequent smear-positive disease. They may thus very likely experience adverse health effects including chronic pulmonary impairment Further research is needed to understand to what extent treatment defaulters contribute to overall transmission of tuberculosis within high-burden communities and whether preventing default is an efficient means for reducing tuberculosis transmission. Further, it needs to be determined whether treatment default contributes to the acquisition and transmission of drug resistance in high-burden communities.


While improvements in genotyping technology have allowed for increased throughput and reduced time and expense, protocols remain hindered by the slow upstream steps of isolating, purifying, and normalizing DNA. Various methods exist for genotyping samples directly through blood, without having to purify the DNA first. These procedures were designed to be used on smaller throughput systems, however, and have not yet been tested for use on current high-throughput real-time (q)PCR based genotyping platforms. In this paper, a method of quantitative qPCR-based genotyping on blood without DNA purification was developed using a high-throughput qPCR platform.The performances of either DNA purified from blood or the same blood samples without DNA purification were evaluated through qPCR-based genotyping. First, 60 different mutations prevalent in the Ashkenazi Jewish population were genotyped in 12 Ashkenazi Jewish individuals using the QuantStudio™12K Flex Real-Time PCR System. Genotyping directly from blood gave a call rate of 99.21%, and an accuracy of 100%, while the purified DNA gave a call rate of 92.49%, and an accuracy of 99.74%. Although no statistical difference was found for these parameters, an F test comparing the standard deviations of the wild type clusters for the two different methods indicated significantly less variation when genotyping directly from blood instead of after DNA purification. To further establish the ability to perform high-throughput qPCR based genotyping directly from blood, 96 individuals of Ashkenazi Jewish decent were genotyped for the same 60 mutations  and resulted in a call rate of 98.38% and a diagnostic accuracy of 99.77%.This study shows that accurate qPCR-based high-throughput genotyping can be performed without DNA purification. The direct use of blood may further expedite the entire genotyping process, reduce costs, and avoid tracking errors which can occur during sample DNA purification. Many advances have been made in the field of genetics, including progress in high-throughput genotyping. The potential turnaround time due to the improved speed of genotyping however, is hindered by the slow upstream step of extracting genomic (g)DNA from blood samples. Manual or automated extraction of gDNA from blood can take at least several hours, without including the time it then takes to normalize each individual sample. Additionally, different volumes of blood are needed for different extraction methods, which are not always obtainable. Commercially available products from multiple vendors now allow for samples to be quickly prepared for genotyping without requiring DNA extraction. For instance, whole blood can be used directly in reactions, and has already been used for gel-based multiplex PCR experiments . With thWhile these commercial methods have been validated for use with smaller throughput systems, such as gels or 96/384-well plates, they have not been tested on the recently developed high-throughput genotyping platforms. Therefore, this study investigates the performance of high throughput PCR-based genotyping directly from blood, without DNA purification. The results of this study will not only be informative for genotyping, but also for transitioning from low-throughput to high-throughput PCR-based experiments in other fields.In order to test the performance of high throughput PCR-based genotyping from blood, we incorporated a multiplex preamplification step of the targeted loci. Genotyping of >12,000 targets in one run was conducted through the use of the TaqMan Sample-to-SNP Kit and the QuantStudio 12K Real-Time PCR System , which is representative of other available high-throughput PCR-based genotyping platforms. The blood and gDNA used in this study was obtained from individuals of Ashkenazi Jewish decent over the age of 18 with patient consent. Three 4 mL lavender tubes of blood were obtained for each patient through the butterfly phlebotomy method, and the blood samples were stored at 10-15°F until needed.The 12 blood samples used in the initial validation had been collected, frozen, and stored over a period of time ranging from 1998-2009. For the purified samples, gDNA was extracted using the QIAamp DNA Blood Maxi Kit  and the concentrations were obtained using a Nanodrop-8000 spectrophotometer . The samples were then normalized to 50 ng/uL. The unpurified samples were processed using the TaqMan Sample-to-SNP Kit protocol as recommended by the supplier (LTI). Two microliters of blood were used for each sample, and a primer pool was made consisting of 64 40X assays diluted 1:200. The 64 assays included 1 assay to detect a SNP on the Y-chromosome for gender identification purposes, 2 assays each for 3 different large deletion mutations [The results from both methods were analyzed separately due to method-based differences observed in clustering and signal intensities. Specifically, since the unpurified DNA underwent a preamplification step while the purified DNA did not, the unpurified DNA had higher signal intensities along both axes when compared to the purified DNA. The same samples were genotyped using both methodologies, however, so a direct analysis could be made between using normalized, purified DNA samples, or unpurified samples consisting of whole blood.CFTR gene to cause Cystic Fibrosis, a nonsense mutation (Y231X) [ASPA gene to cause Canavan disease, a small deletion (delR608) [SMPD1 gene to cause Niemann-Pick type A, and two frameshift mutations . All of the assays were designed with the wild type allele having the VIC probe and the minor allele having the FAM probe.Of the twelve samples, five were known to be heterozygous carriers for one mutation each. The five mutations included a splicing mutation 1717-1G>T) [17-1G>T [ (Y231X)  in the AdelR608)  in the Sns (84GG  and 1278ns (84GG ) in the A large-scale blinded study was then done on 96 blood samples in duplicate to further assess the validity of high-throughput genotyping directly from blood. The blood samples were processed using the TaqMan Sample-to-SNP Kit protocol as mentioned above and consisted of 58 male and 38 female samples collected, frozen, and stored over a period of time ranging from 1992-2011  or absence (non-amplification) of the SNP. Since samples do not always amplify during PCR-based reactions, a better method for gender identification would be to target the single base differences in the amelogenin genes as previously described . This meAn important aspect of this study included using blood samples that had previously been frozen and stored. It is the common practice of many laboratories to store frozen blood samples for later use. While these samples are still viable for extracting gDNA once thawed , we wantUsing samples directly for high-throughput genotyping without DNA extraction will continue to be investigated by our lab. In the future, reference samples will be incorporated during the analysis to further aid in correctly genotyping samples, with the hope that eventually this methodology will be used to perform routine carrier screening in a truly high-throughput fashion. Potential benefits of using blood directly to genotype include saving time and money, while improving sample information tracking by avoiding purification steps where samples are transferred between multiple tubes. Since this study has shown that DNA purification is not necessary for accurate PCR-based genotyping, the direct use of blood can therefore be used to further expedite the entire genotyping process, especially when combined with a high-throughput genotyping platform.The data sets supporting the results of this article are included within the article and its additional file.gDNA: genomic DNA; qPCR: Real time PCR.The authors declare that they have no competing interests.AF carried out the study and drafted the manuscript. JS carried out the study. CJ participated in the design of the study. NT conceived of the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.Figure S1. Methodology Comparison. A. Allelic discrimination plots depicting genotyping results using purified DNA. B. Allelic discrimination plots depicting genotyping results using blood directly for the same samples genotyped in A. The wild type allele used the VIC probe (y-axis) while the minor allele used the FAM probe (x-axis).Click here for file


A major atherogenic process is the formation of lipid-loaded macrophages in which a breakdown of the endolysomal pathway results in irreversible accumulation of cargo in the late endocytic compartments with a phenotype similar to several forms of lipidosis. Macrophages exposed to oxidized LDL exihibit this phenomenon in vitro and manifest an impaired degradation of internalized lipids and enhanced inflammatory stimulation. Identification of the specific chemical component(s) causing this phenotype has been elusive because of the chemical complexity of oxidized LDL.Atherosclerosis starts by lipid accumulation in the arterial in situ and (much faster) by intracellular aldehyde oxidizing systems to cholesteryl hemiesters. We show that a single cholesteryl hemiester incorporated into native, non-oxidized LDL induces a lipidosis phenotype with subsequent cell death in macrophages. Internalization of the cholesteryl hemiester via the native LDL vehicle induced lipid accumulation in a time- and concentration-dependent manner in “frozen" endolysosomes. Quantitative shotgun lipidomics analysis showed that internalized lipid in cholesteryl hemiester-intoxicated cells remained largely unprocessed in those lipid-rich organelles.Lipid “core aldehydes" are formed in oxidized LDL and exist in atherosclerotic plaques. These aldehydes are slowly oxidized The principle elucidated with the present cholesteryl hemiester-containing native-LDL model, extended to other molecular components of oxidized LDL, will help in defining the molecular etiology and etiological hierarchy of atherogenic agents. A “fatty streak" that reflects deposits of lipid in the intima is the first visible sign of this pathology  in the arterial via the LDL-receptor are delivered to lysosomes where their cholesteryl esters are hydrolyzed by acid hydrolases to free cholesterol that is then either exported from the cell or re-esterified in the endoplasmic reticulum and stored in the cytosol in lipid storage droplets via a large number of receptors that are not particularly specific to LDL via macropinocytosis Normally, LDL endocytosed by cells in vivo in the artery wall and stimulate endothelial cells to produce pro-inflammatory molecules that recruit monocytes and promote their differentiation to macrophages. The macrophages then internalize the Ox-LDL, are unable to metabolically handle it, and begin to accumulate ingested cargo irreversibly in the endolysosomal compartment in a manner similar to that seen in lipid storage diseases The “oxidative-modification of LDL" hypothesis via the action of intracellular aldehyde oxidizing systems, they will be oxidized to stable cholesteryl hemiesters of the corresponding bi-acids as shown in in vitro as components of Ox-LDL 2-glycoprotein 1, a major antigen for antiphospholipid antibodies present in patients with antiphospholipid syndrome in which high serum levels of the complexes of Ox-LDL with β2-glycoprotein 1 are associated with arterial thrombosis Ox-LDL contain a myriad of lipid oxidation products with biological activity. Cholestryl linoleate and cholesteryl arachidonate, the predominant cholesteryl esters with more than one double bond in LDL, produce several aldehydes upon oxidation: some  are quite water-soluble while others are less polar and have variable degrees of partitioning between lipid and aqueous phases in vitro to oxidize LDL is debatable, and the complexity of the product with regard to the oxidized components makes these models of Ox-LDL difficult to work with. This is particularly applicable to comparisons of studies from different laboratories since the detailed chemical composition of Ox-LDL is almost never reported and preparations isolated from tissues vary greatly among laboratories , in Nat-LDL. Specifically, we examined the involvement of Chs in the formation of a lipidosis phenotype and in cell death when macrophages were exposed to Chs-loaded Nat-LDL (Chs-LDL). We show that Chs is sufficient to induce a lipidosis phenotype and apoptotic cell death, two characteristic features of Ox-LDL. We also show that Chs-LDL lead mainly to lipid over-accumulation in endolysosomal structures, not lipid-storage organelles. Our results suggest that in vitro experimental Ox-LDL models have been used to examine the molecular etiology of atherogenic lipidosis in macrophages and, consequently, elucidate the origin of atherogenesis. However, the multitude of oxidation products in Ox-LDL makes it difficult to establish etiological hierarchies. To avoid this complication and study the biological effects of a specific family of compounds that may be formed upon oxidation of LDL, we generated a new model of LDL enriched specifically with Chs as a model cholesteryl hemiester. Cholesteryl-4-oxobutyrate, a precursor of Chs, is one of the “core aldehyde" products of the peroxidation of LDL in vitro under conditions of oxidative stress, and have been identified as ligands for the plasma protein β2GPI 2GPI complexes in vivo seem to be correlated with acute coronary syndromes Several 3H-Chs and by assessing the LDL charge by electrophoretic mobility in an agarose gel. Chs-LDL were generated by incubating Nat-LDL with Chs-containing POPC liposomes (with a Chs/POPC molar ratio of 70∶30) at different LDL∶liposome ratios. After incubation, the LDL particles were re-isolated by ultracentrifugation see  to elimiin vitro, it must be emphasized here that Chs-LDL particles are only a model for cholesteryl hemiester-containing LDL without any other alteration of the chemical constitution of the particles. We emphasize here that cholesteryl hemiesters have been largely ignored in the literature as potential causes of atherogenesis.Since this work is an attempt to present a new approach, and to establish a proof of concept, to study the molecular etiology of lipidosis Macrophages and other sub-endothelial cells suffering from lipidosis are a defining characteristic of atherosclerotic plaques and are believed to be one of the first stages in atherogenesis. Ox-LDL are known to induce lipidosis in macrophages and subsequently cause apoptotic cell death due to uncontrolled uptake of lipid-rich particles combined with putative blockage of any one (or more) of several lipid processing reactions. Qualitatively, the process is morphologically similar to phenotypes seen in several lipid-storage pathologies intima range from about 0.7 to 2.7 mg/ml The effect of Ac-LDL, Chs-LDL and Nat-LDL concentration on total volume of lipid deposits per cell as a function of time is shown in 2 and 3 µm2 in cells incubated with 400 µg/ml of Ac-LDL and Chs-LDL, respectively.Another striking difference between the lipid-rich structures in macrophages incubated with Ac-LDL and Chs-LDL was their average cross sectional areas – 0.8 µm50, Chs concentration at which cell viability was 50% of the control, was determined for each exposure time  Thus, RAW cells exposed to Chs-LDL accumulate neutral lipids in late endosome/lysosome structures , Chs-LDL, and Chs-POPC liposomes with similar total Chs concentrations. The intracellular radioactivity was examined as a function of incubation time . Surprisvia CD204 and SR-BI receptors, but not CD36, contributes in a small measure to Chs internalization via the Ac-LDL vehicle To further clarify this apparent vector-independent Chs uptake, we examined the effect of blocking scavenger receptors in RAW cells that are known to play a role in the uptake of modified LDL, namely, CD36, SRA (CD204) and SR-BI Because, Chs gets into the cells mainly by passive diffusion and Chs-LDL appear to be poor substrates for lysosomal enzymes  we hypotThe last question we addressed was how macrophages handled the accumulated lipid in the various cases examined in this work. Reversibility of lipid accumulation in RAW cells was assessed by pulse-chase experiments . In cellThese results suggest that lipid accumulation in endolysosomes within the cells exposed to Chs  is an irintima is generally accepted to be one of the initial steps of atherogenesis in the arterial wall. These cells that originate atheromata have a defective cholesterol homeostasis that results in irreversible accumulation of lipid in endolysosomes, a property that is characteristic of many endolysosomal lipid storage diseases. Why exactly this happens is debated but there seems to be a consensus that the process involves defective post-internalization handling by macrophages (and other cells) of trapped and physico-chemically modified LDL in the subendothelial space.The induction of lipidosis in macrophages in the 9) chain dicarboxylic acid. Several ω-oxoester precursors of hemiesters (of C4 through C9 diacids) of cholesterol have been identified in Ox-LDL 2-glycoprotein-1, and shown to be involved in the formation of atherogenic complexes of this protein with Ox-LDL in vivo for atherogenesis.One of the chemical modifications of LDL that has received much attention is the oxidative modification of their linoleic and arachidonic acid-containing components. Oxidative breakdown of either of these fatty acids produces a very large number of products many of which are aldehydes. In this category of products 4-hydroxynon-2-enal (HNE) and malondialdehyde (including its protein conjugates) have been most extensively studied. But it is also known that HNE is very rapidly  converted by cells to other products, the principal one being 4-hydroxynon-2-enoic acid Ox-LDL are known to induce lipidosis with irreversible endolysosomal accumulation of lipid from macrophages in vitro 4 diacid) model for the several hemiesters of cholesterol that can be formed in Ox-LDL. These hemiesters are all amphiphilic, have a negative charge at physiological pH and can be expected to partition favorably into cell membranes; they equilibrate through passive diffusion between the Ox-LDL (where they are formed) and the aqueous (extra- and intracellular) phases, as well as all membranes of neighboring cells. Cholesteryl hemiesters, or for that matter, their precursor ω-oxoacid esters of cholesterol, have been mostly ignored as atherogenic agents in the literature although the other scission product of cholesteryl linoleate, HNE, has received abundant attention. The methodology we describe here may be profitably used to study the ability of several other components of Ox-LDL (isolated or in controlled combinations) to induce lipidosis with irreversible endolysosomal lipid accumulation in macrophages and other sub-endothelial cells. The model may, in principle, be useful in studying detailed kinetics of the phenomenon as well as to study synergistic and/or antagonistic processes among the chemical constituents of oxidized, or otherwise modified, LDL. We have previously described strategies for enriching lipid aggregates (including LDL) with different kinds of amphiphiles In consideration of the above, we decided to develop a model Nat-LDL particle enriched in a single chemical constituent of Ox-LDL that could possibly be responsible for the induction of lipidosis. We demonstrate this principle here with native non-oxidized LDL enriched in Chs, a cholesteryl hemiester (of a CThe effects of Chs on RAW cells may be summarized as follows: 1) Formation of lipid loaded cells with lipid accumulation in the endolysosomal compartment; 2) Incapacity of affected cells to metabolize (hydrolyze and re-esterify) internalized cholesteryl esters; 3) Irreversible and uncontrolled lipid accumulation in the endolysosomal compartment; and 4) Apoptotic cell death.via passive diffusion and equilibration between the LDL particle and its immediate environment . Chs is an amphiphile which, due to its cholesterol moiety, partitions very favorably into membranes. The simplest effect that can be imagined is an increase in membrane order, similar to the effect of cholesterol +-ATPase which is responsible for acidification of lysosomes We have no data that informs us as to exactly why Chs has these effects. The reasons may be complex but we shall speculate on this theme. We have shown that the internalization of Chs is largely a vehicle-independent process, probably occurring mostly Clearly the effects observed by us are not simply due to ingestion of a large amount of cholesteryl esters by the cell – the cholesteryl esters of Ac-LDL, which are internalized more rapidly than Chs-LDL, are processed normally and do not create a pathological state. The pathological state is caused by the Chs in Chs-LDL. Our results  indicateWe recognize that the Chs concentrations to which the RAW cells were subjected in this work may be much higher than can be accounted for under physiological conditions. However, the spontaneous oxidation of Nat-LDL in cell culture media imposes limits on exposure times and, therefore, requires higher doses. A more detailed study of the dose and exposure-time dependence of the effects we report here may be warranted in the future.Finally, we note with particular interest, that whereas the large majority of the RAW cells subjected to Chs exposure died apoptotically, a small subset, representing between 5 and 10% of the population, survived and seemed to accumulate lipid progressively and irreversibly in the endolysosomal compartment. This observation may be related to macrophage heterogeneity 3H]-Colesterol was from GE Healthcare. The other chemicals used were of analytical grade from local sources.Oil-red, Cholesteryl hemisuccinate, Dextran  were obtained from Sigma. PO-PC and cholesterol were purchased from Avanti Polar Lipids . -Chs was determined after rinsing macrophages with DMEM with 0.5% BSA at pH 3.4 for 10 min on ice, three further rinses with PBS, and a final rinse in distilled water. Macrophages were scraped off into 1 ml distilled water. Then cell samples were assayed for 3H radioactivity and for cellular protein.After incubating the macrophages with 3H-Chs-(Ac-LDL), 3H-Chs-LDL, or 3H-Chs-POPC liposomes (45∶55) with equivalent amounts of Chs were added to cells. The cells were then shifted to 37°C for 3 h (in presence of the inhibitory antibodies). After incubation the cells were treated as described above and the radioactivity and the cell protein quantified.For the experiments with the inhibitory antibodies the cells were incubated at 4°C for 15 min with 10 µg/ml of anti-CD36, anti-SR-BI, anti-CD204 and their respective antibody controls; after which 300 µg/ml of The protein content of cell extracts was measured by the bicinchoninic acid  assay using bovine serum albumin as a standard.t-test (two-tailed). A p value of <0.05 was considered to be statistically significant.Results are expressed as the means ± standard deviations (S.D.). Statistical significance was assessed by the Student Figure S1Chs-LDL are poorly degraded within the lysosomal structures. (a) Macrophages were incubated for 24 h with 300 µg/ml of Nat-LDL (lane 1), Ac-LDL (lane 2) or Chs-LDL (lane 3). In lane 4 lipid extracts of cells incubated with Chs∶POPC liposomes (45∶55) were loaded. At the end of the incubation time the lipids were extracted and resolved by TLC as in (PDF)Click here for additional data file.Methods S13H-Chs. Lipid extraction and preparative TLC.Synthesis of  Intracellular Chs hydrolysis.(PDF)Click here for additional data file.Table S13H-Chs in RAW cells.Hydrolysis and re-esterification of cholesterol derived from  RAW cells were treated for 24 and 48 h with 3H-Chs-POPC liposomes (45∶55). The compounds were separated by TLC prior to measurement of radioactivity.(PDF)Click here for additional data file.


In April 2009, the most recent pandemic of influenza A began. We present the first estimates of pandemic mortality based on the newly-released final data on deaths in 2009 and 2010 in the United States.We obtained data on influenza and pneumonia deaths from the National Center for Health Statistics (NCHS). Age- and sex-specific death rates, and age-standardized death rates, were calculated. Using negative binomial Serfling-type methods, excess mortality was calculated separately by sex and age groups.In many age groups, observed pneumonia and influenza cause-specific mortality rates in October and November 2009 broke month-specific records since 1959 when the current series of detailed US mortality data began. Compared to the typical pattern of seasonal flu deaths, the 2009 pandemic age-specific mortality, as well as influenza-attributable (excess) mortality, skewed much younger. We estimate 2,634 excess pneumonia and influenza deaths in 2009–10; the excess death rate in 2009 was 0.79 per 100,000.Pandemic influenza mortality skews younger than seasonal influenza. This can be explained by a protective effect due to antigenic cycling. When older cohorts have been previously exposed to a similar antigen, immune memory results in lower death rates at older ages. Age-targeted vaccination of younger people should be considered in future pandemics. In April 2009, a novel strain of influenza A/H1N1 emerged in Mexico, rapidly spreading to the United States In this article, we analyze final mortality data for the United States which were released in August 2012. This is the first analysis of the complete mortality record of influenza for 2009 and 2010. Our age- and sex-specific analysis incorporates data from 1959–2010, and pneumonia and influenza deaths were analyzed together All the data used in this study are fully in the public domain. We obtained data on number of deaths, by cause, from the mortality detail files of the National Center for Health Statistics (NCHS) Rates  were calculated using these death counts in the numerator, and exposure data (person-years at risk) from the Human Mortality Database Excess mortality was calculated using modified Serfling-type methods Count regression models produce estimates that are broadly comparable to classic Serfling methods Upper and lower bounds were calculated from jacknifed standard errors As The estimates from The public health concern about influenza pandemics stems, above all else, from the potential to kill millions, as in the 1918–19 pandemic Seasonal influenza typically kills with an old-age mortality pattern: the cross-hatched bars (1999–2008 average) of Overall, 2009 had about 2,438 pneumonia and influenza excess deaths, and 2010 had about 196. Our estimated total excess deaths is 2,634. The bulk of pneumonia and influenza mortality typically occurs above age 60, but when the pandemic strain of influenza H1N1 displaced other circulating strains After reemergence in 1977, influenza A/H1N1 spread in children and young adults because of its remarkable antigenic similarity to the pre-1957 H1N1 strains This study is not without limitations. We use only publicly-available data and therefore the time resolution is monthly. Although we employed negative binomial regression as an update to classical Serfling analysis, the present study shares the limitations of any technique that analyzes mortality data by itself without bringing in viral surveillance co-variates One of the biggest challenges of pandemic preparedness is rapid formulation and manufacture of a strain-specific vaccine


Caenorhabditis elegans is an ideal organism for studying neural plasticity and animal behaviors. A total of 302 neurons of a C. elegans hermaphrodite have been classified into 118 neuronal groups. This simple neural circuit provides a solid basis for understanding the mechanisms of the brains of higher animals, including humans. Recent studies that employ modern imaging and manipulation techniques enable researchers to study the dynamic properties of nervous systems with great precision. Behavioral and molecular genetic analyses of this tiny animal have contributed greatly to the advancement of neural circuit research. Here, we will review the recent studies on the neural circuits of C. elegans that have been conducted in Japan. Several laboratories have established unique and clever methods to study the underlying neuronal substrates of behavioral regulation in C. elegans. The technological advances applied to studies of C. elegans have allowed new approaches for the studies of complex neural systems. Through reviewing the studies on the neuronal circuits of C. elegans in Japan, we will analyze and discuss the directions of neural circuit studies.The nematode  Understanding the mechanisms underlying neural operation and processing is a major goal in neuroscience. One approach is to study a simple neural circuit, in which neurons and neural pathways can be identified and diagrammed for analysis. Another approach is the application of molecular genetic techniques to reveal the molecular components that govern the neural functions and clarify the site of action of molecules in brain .Caenorhabditis elegans is an ideal organism for studying neural circuits and related behavior, since it completely fulfills the above-mentioned criteria. The nervous system of C. elegans consists of only 302 neurons and the entire neuronal system of C. elegans is composed of approximately 5,000 synapses and 600 gap junctions, as revealed by electron microscopic analysis . Several studies have shown that C. elegans is not only capable of non-associative learning and short-term memory, but also capable of associative learning and long-term memory . Pre-exposure to butanone with food did not change the degree of chemotaxis to benzaldehyde, suggesting that this enhancement of chemotaxis was odorant-specific. The enhancement of chemotaxis to butanone did not require serotonin, which is responsible for food signaling, despite the essential role of food during the conditioning.Butanone and benzaldehyde are attractive odorants that are sensed by AWC olfactory neurons . TorayamFigure 2B). The first mutant, olrn-1, had a mutation in the gene encoding a novel transmembrane protein. Interestingly, olrn-1 expression was strictly required in AWC neurons for the proper butanone enhancement. The left and right AWC neurons develop distinct sensory properties through an unusual stochastic lateral signaling interaction  genes  genes . BBS is S) genes . The C.  neurons . Indeed,ry cilia . The expForgetting is the process of eliminating unnecessary or excessive information in the brain, enabling animals to obtain new information from their continuously changing environment. Despite the importance of forgetting, the neural and molecular bases of forgetting are almost unknown.C. elegans shows attractive responses and adaptation to odorants sensed by two pairs of olfactory neurons, the AWA neurons and the AWC neurons . C. elegans animals grown with food were exposed to diacetyl in foodless conditions for 1.5 h. Then, the animals were re-grown on food-rich plate for a certain period of time before a chemotaxis assay was performed . Wild type animals pre-exposed to diacetyl for 1.5 h recovered normal chemotaxis to diacetyl within 4 h of exposure to food. In contrast, tir-1 mutants isolated by forward genetics screen showed long-lasting adaptation: they gradually recovered the chemotaxis to diacetyl over about 25 h . Calcium imaging revealed that the AWA response to diacetyl was suppressed during the adaptation period and recovered after 4 h in wild type animals, whereas the AWA response to diacetyl was still suppressed after 4 h in tir-1 mutants. The sensory perception and the adaption to diacetyl were normal in tir-1 mutants, suggesting that tir-1 mutants are defective specifically in the process of forgetting. Notably, tir-1 encodes a Toll/interleukin-1-resistance domain protein that is homologous to the mammalian adaptor protein SARM, which is known to be expressed in the brain.Olfactory adaptation is the plastic behavior in which animals pre-exposed to an odor in the absence of food show decreased response to the odor compared with naive animals.  neurons . Inoue eC. elegans, the TIR-1-mediated p38 mitogen-activated protein kinase (MAPK) pathway is known to regulate the asymmetric cell fate decision of AWC neurons. It also regulates the innate immune response. The UNC-43(CAMKII)-TIR-1-NSY-1(MAPKKK)-SEK-1 (MAPKK) pathway is required for the determination of AWC cell fate, whereas the TIR-1-NSY-1-SEK-1-PMK-1 (MAPK) pathway is required for the innate immune response . The jnk-1 mutants displayed forgetting defects, thus, the process of forgetting diacetyl is regulated through the TIR-1/JNK-1 pathway, which is partly overlapping but distinct from the pathways for AWC-asymmetric cell fate and the innate immune response. The TIR-1/JNK-1 pathway is critical for the forgetting process of AWC-sensed odor and of salt chemotaxis learning, suggesting the general role of the TIR-1/JNK-1 pathway in forgetting.In response . Inoue etir-1 cDNA into tir-1 mutants under the AWA-specific promoter did not rescue the phenotype, but under the AWC-specific promoter rescued the defects. Expression of sek-1 cDNA into sek-1 mutants under the AWC-specific promoter also rescued the forgetting defects. Likewise, the expression of the dominant negative forms of sek-1 and jnk-1 only in the AWC neurons mimicked the forgetting-defective phenotype. These results suggest that the TIR-1/JNK-1 pathway in AWC neurons is sufficient and necessary for the forgetting process. Two pieces of evidence support the notion that the AWC cell itself plays an important role in the forgetting process of AWA neurons. First, ceh-36 mutants, in which functional AWC neurons are undifferentiated because of developmental errors, exhibited forgetting defects  mutants, a special allele of the tir-1 gene that retains AWC asymmetry, also exhibited forgetting defects. AWC neurons are critical for forgetting the adaptive process of AWA-sensed odor, and the TIR-1/JNK-1 pathway in the AWC neurons is essential for the forgetting process .The neurons responsible for the process of forgetting diacetyl have been identified. Expression of  defects . Second, defects . The brergetting ; and tirpkc-1 in AWC neurons rescued the forgetting defects in tir-1 mutants, although it does not affect the chemotaxis and adaptation to diacetyl, suggesting that the TIR-1/JNK-1 pathway regulates neurosecretion from AWC neurons. Reciprocally, the expression of TetX (tetanus toxin light chain), an inhibitor of synaptic transmission in AWC neurons, caused wild type animals to exhibit the forgetting defects. Thus, AWC neurons may send a forgetting-accelerating signal to AWA neurons through secretion molecules. What induces AWC neurons to release the forgetting signal? The sensory perception of AWC neurons is not involved in forgetting, because tax-4 mutants defective in AWC sensory signaling showed normal forgetting behavior . The enhancement of the touch response in C. elegans has also been reported, yet the molecular and neural bases for this are unknown  and acidic pH as an US, and then conducted spaced training and massed training. Spaced training consists of repeated trials with an inter-trial interval (ITI), whereas massed training consists of repeated trials without an ITI. The memory after the spaced training was retained for 24 h, whereas the memory after the massed training lasted only 3 h. Consistent with the theory for touch response  transcriptional factor only affect long-term memory.response , C. eleg2+ ion is aversive metal sensed by ASH and ADL sensory neurons . When C. elegans encounters the Cu2+ barrier during their migration toward diacetyl, the balance between concentrations of diacetyl and Cu2+ regulates the behavioral decision whether they go straight toward diacetyl or withdraw .Decision-making is the cognitive process by which animals select one action among several different choices. In order to study behavioral decision from two conflicting choices and the integration of two different sensory cues, an interaction assay system was developed . Diacety neurons . The atthen-1 gene, which encodes a secretory protein with an LDL (low-density lipoprotein) receptor motif, regulates sensory integration and decision-making. In the interaction assay, hen-1 mutants showed a weaker tendency to cross the Cu2+ barrier when approaching the attractive odorant diacetyl. Since hen-1 mutants show a normal attractive response to diacetyl and aversion from the Cu2+ ion, the HEN-1 protein likely plays a role in decision-making in behavioral choice . The cell non-autonomous function of the HEN-1 protein suggests that HEN-1 acts as a secreted molecule. Secretion of HEN-1 from AIY interneurons is crucial because ttx-3 mutants .The jeb protein in tor Dalk . SCD-2 i mutants . These rces cGMP . It was  channel  that fun2+ form synapses with AIA neurons. ASH neurons are known to be glutamatergic, and the glutamate-gated chloride channel GLC-3 is known to be functional in AIA neurons . Although AIA neurons send synaptic outputs to many neurons, a major synaptic target of AIA neurons are AIB interneurons that are known to regulate a reversal of movement: removal of an odor sensed by AWC neurons activates AIB neurons to produce a reversal behavior, which reorients the animals to the odor source . Further analysis, such as clarification of the physiological properties of AIA neurons, identification and analysis of other molecules, and clarification of the relationship between these molecules will shed light on the decision-making process at the molecular and circuit levels.AIB interneurons receive synaptic inputs from ASH and ADL neurons, both of which sense aversive stimuli, including Cuilon/eta  caused glearning , indicatCaenorhabditis elegans strains isolated from natural environments all over the world are categorized based on behavioral properties into two group: social and solitary strains. Social strains show the aggregation of animals on the boundary of food where oxygen concentration is low. The standard laboratory strain of C. elegans is the Bristol type, which was isolated in England and exhibits the solitary phenotype . On the other hand, the overexpression of snet-1 weakened the tendency toward dispersion. The nep-2 gene encoding the extracellular peptidase neprilysin was identified as the negative regulator that controls the activity of SNET-1. In nep-2 mutants, the degradation of SNET-1 peptide is inhibited and the accumulated SNET-1 suppressed dispersion behavior. Taken together, the information on population density is transmitted through external pheromone and endogenous peptide signaling, thereby assuring behavioral plasticity that may be important for the survival of species  showed that ASEL neurons responded to the addition of salt, whereas ASER neurons responded to the removal of salt . The insulin-like signaling pathway is well known to regulate dauer formation  and aging in C. elegans . elegans . MutantsSuppression of synaptic release was supported by the finding that the activity of AIB neurons, one of the downstream interneurons of ASER neurons, is downregulated after salt learning . The molcasy-1. CASY-1 is a transmembrane protein carrying two tandem cadherin domains and an LG/LNS domain in the ectodomain. CASY-1 is an ortholog of calsyntenins  that is reported to be involved in episodic memory performance in humans .n humans . The casCaenorhabditis elegans associates past growth temperature with food. In its natural habitat, C. elegans likely adapts to the fluctuating temperatures in soil in order to stay near food sources. We can observe this behavior as thermotaxis in the laboratory . Growth temperature-shift experiments indicated that the acquisition of a new temperature memory requires 2–4 h. Dynamic alternation of temperature preference is also induced by starvation. Growth without food at a certain temperature for several hours induces animals to disperse or avoid the past growth temperature .boratory . After aC. elegans, the environmental temperature is sensed by AFD and AWC sensory neurons, and then thermal signals are transmitted to the downstream interneurons AIY, AIZ, and RIA. The AIY-mediated neural pathway is responsible for thermophilic movement, whereas the AIZ-mediated neural pathway is responsible for cryophilic movement. The counterbalanced regulation of the activities of AIY and AIZ neurons is thought to be essential for thermotaxis. The RIA interneurons integrate signals from both upstream interneurons AIY and AIZ neurons. Consequently, the outcome of neural computations in the core thermotaxis circuit regulates the body wall muscles, thereby controlling the worm’s ultimate behavior .The simple neural circuit involved in thermotaxis is the perfect subject for the study of the functional connectome. In 2+ imaging, where the response of AFD neurons to warming corresponded to their past growth temperature . These results suggest that the balanced regulation through these two monoamines in interneurons is a key process for thermotactic plasticity. It is intriguing to ask how the antagonistic behavioral regulation through serotonin and octopamine is related to the antagonistic neural regulation between AIY and AIZ neurons .We propose that the neural circuit for thermotactic plasticity is amazingly similar to two functional parts of the human brain: the cerebral cortex and basal ganglia  is a particularly challenging problem. Using the voltage-sensitive fluorescent protein VSFP 2.42  of worms on agar plates using several hours of data. The heads and tails of tracked worm are also identified from acquired images using their algorithm , designed to cause a blue shift in the action spectrum, is expressed in C. elegans neurons, locomotory arrest can be elicited by blue light, proving that C. elegans locomotion can serve as a convenient assay for in vivo evaluation of new optogenetic tools  and on a higher plant  is a novel recently developed system that uses an infrared (IR) laser radiating at 1480 nm, a wavelength at which the absorption coefficient of water is about 10t 440 nm . The muc neurons . By chooin vitro study using a polyacrylamide gel tissue model has suggested that temperatures at the irradiation focus change very quickly in response to IR irradiation, and that the area with temperature shifts exceeding 20°C was essentially confined to a vertically extended ellipsoidal area 7 μm along the x–y axes  and full recording of all neuronal activity in the near future will be an important boost for the brain activity map project. Given this situation, C. elegans neuroscience is becoming more valuable than ever.As we have reviewed above, studies using an brain , 2013. TThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The transcriptome from leaf, root, and young panicle at three developmental stages was comparatively analyzed combined with bioinformatics exploring drought stress related cis-elements.Rice is highly sensitive to drought, and the effect of drought may vary with the different genotypes and development stages. Genome-wide gene expression profiling was used as the initial point to dissect molecular genetic mechanism of this complex trait and provide valuable information for the improvement of drought tolerance in rice. Affymetrix rice genome array containing 48,564 GA2ox, SAP15, and Chitinase III, were regulated in a reciprocal way in two tissues at the same development stage. A total of 261 transcription factor genes were detected to be differentially regulated by drought stress. Most of them were also regulated in a tissue- or stage-specific manner. A cis-element containing special CGCG box was identified to over-present in the upstream of 55 common induced genes, and it may be very important for rice plants responding to drought environment.There were 5,284 genes detected to be differentially expressed under drought stress. Most of these genes were tissue- or stage-specific regulated by drought. The tissue-specific down-regulated genes showed distinct function categories as photosynthesis-related genes prevalent in leaf, and the genes involved in cell membrane biogenesis and cell wall modification over-presented in root and young panicle. In a drought environment, several genes, such as cis-element for drought responsiveness, is a very useful resource for the functional dissection of the molecular mechanism in rice responding to environment stress.Genome-wide gene expression profiling revealed that most of the drought differentially expressed genes (DEGs) were under temporal and spatial regulation, suggesting a crosstalk between various development cues and environmental stimuli. The identification of the differentially regulated DEGs, including TF genes and unique candidate  Rice is sensitive to drought stress because it is acclimated to either rain-fed or fully irrigated fields. The effect of drought on rice plants considerably varies with genotypes, different developmental stages, and degree and duration of drought stress. Relatively, rice plants are less affected by water deficit at the seedling stage, although drought stress at the vegetative stage does result in reduced height, fewer tillers, and smaller leaf area. However, rice is highly sensitive to water deficit at the panicle initiation and booting stages ,2.Drought tolerance (DT) is a complex trait both genetically and physiologically. Developing DT varieties by breeding is the major strategy for reducing rice yield losses caused by drought. However, to date, few successes have been achieved in this effort because plant DT is a typical quantitative trait influenced by many different genes . Genome-Drought stress causes a wide range of physiological and biochemical responses in plants. These responses include reduced stomatal conductance and photosynthesis, and accumulation of osmolytes and proteins in cells . A numbeWith the development of molecular technology and advancement in nanotechnology, DNA microarrays have been devised as a standard strategy for the global analysis of plant gene expression. Microarrays can simultaneously detect thousands of targets in a high throughput manner, and thus their use has enormously expanded to cover all kingdoms of living organisms. The availability of complete genome sequences and of huge EST collections allows the development of different microarray platforms in rice. Several biological processes and important traits of rice have been analyzed using cDNA microarray or whole genome array including salt-responsive genes by cDNA microarray analysis , geneticTo achieve a more comprehensive understanding of the global spatial and temporal gene expression patterns of rice in response to drought stress, we performed a genome-wide gene profiling analysis using a unique drought tolerant rice line and an Affymetrix whole gene array set. Three tissues  at three developmental stages under drought stress and control conditions were used to profile their gene expression level. A temporal and spatial gene regulation pattern responsive to drought stress was primarily revealed in our study.7 line derived from a cross between two DT IR64 introgression lines (ILs), DGI 187 and DGI 74  stage, panicle elongation stage, and booting stage. Plants were stressed until the leaf became fully rolled at noon (measured leaf relative water content was 65%-75%). It took three and two days for drought stress to become apparent at the tillering and panicle elongation stages and at booting stage, respectively. Both leaf and root samples were collected for the first two stages, and leaves and young panicle samples were collected at the booting stage. Three biological replicates  were prepared for microarray analysis. After collection, samples were snap frozen in liquid nitrogen and kept in a -80°C freezer for total RNA extraction.japonica and 1,260 indica sequences. Hybridization was processed at 45°C, with rotation for 16 h (Affymetrix GeneChip Hybridization Oven 640). Chips were then washed and stained in the Affymetrix Fluidics Station 450 and then scanned using the Affymetrix Gene Chip Scanner 3000. All experiment steps were conducted in CapitalBio Corporation in Beijing.RNA preparation was conducted following the instructions of Affymetrix . Briefly total RNA was extracted from liquid nitrogen frozen sample using TRIZOL reagent according to the instruction, and then purified and concentrated using RNeasy MinElute Cleanup kit  and an on-column DNase treatment as recommended by Affymetrix. The following steps were then processed in CapitalBio Corporation, Beijing. Total RNA of 2 μg was used for synthesizing ds cDNA. Biotin-tagged cRNA was generated from an in vitro transcription reaction using MessageAmp™II aRNA Amplification Kit and then fragmented into 35-200 bases in length according to the Affymetrix protocol. The resulting cRNA was then hybridized to the Affymetrix rice genome array (Affymetrix) containing 48,564 GeneChip Operating Software (GCOS1.4) was used to analyze the hybridization data. The scanned images were first examined by visual inspection and then processed to generate raw data saved as CEL files using the default setting of GCOS1.4. We used dChip software to perform invariant-set normalization according to the dChip user's manual. The whole set of original microarray data has been deposited in NCBI's Gene Expression Omnibus and can be freely accessed through GEO Series number GSE26280.t test of P value less than 0.05 based on three independent biological replicates. The DEGs were performed in complete linkage hierarchical clustering analysis using the TIGR MeV 4.2 software http://www.tm4.org.For comparison analysis, two classes of unpaired methods in the Significant Analysis of Microarray (SAM) software were applied to identify differentially expressed genes (DEGs) between the drought stress sample and the control sample. As there is no fixed standard threshold between significant and non-significant differential gene expressions, we identified the DEGs using the empirical criteria of more than five-fold change and significant http://bioinfo.capitalbio.com/mas/). Significance analysis of GO was performed by the gene enrichment based on hypergeometric distribution finished by Fisher or Chi-sequare test.The putative function of each DEG corresponding to the probe set on the chip was predicted by Affymetrix annotation combined with TIGR definition and NCBI database. GO analysis was performed by a Molecule Annotation System  and the known ABRE motifs were located in the promoter regions (-10 to -1000 bp upstream of the start codon) and compared with the promoter regions of all control genes .A Weeder program  was usedhttp://frodo.wi.mit.edu/. An Actin gene was used as internal control. RT-PCR-amplified products were sequenced, and 100% homology to the target sequences was confirmed. PCR reaction was performed using the same RNA samples used for the microarray analysis. The first-strand cDNA was obtained from 1 μg of total RNA in a 50 μl reaction mixture, and 1 μl of synthesized cDNA was used as template for PCR reaction . All assays were performed in triplicate. After gel electrophoresis, we measured the intensity of each band and normalized the data using the beta actin bands of each well, respectively.Several genes with special tissue-specific or stage-specific DEGs were selected to confirm the expression level of microarray results using RT-PCR. The sequences corresponding to the genes were obtained from the rice genome sequences database (TIGR). The sequences of exons from genes were used to design the RT-PCR primers using the Primer 3 software The objective of this study is to genome-wide profile the expression of genes in rice in a drought environment. To simulate natural growth conditions, rice plants were cultured in the glasshouse. Turface in the growth tubes was used to support plants for the easy and quick collection of root samples under drought treatment and control conditions. This simulated drought strategy is quite different from that previously used, such as hydroponic cultured in PEG solution  or air-djaponica and indica, respectively). Among these, 18,976 (38.1%) to 23,068 (46.4%) genes were detected to be expressed in each of the six samples under stressed or control environments  in at least one of the six samples. A total of 1154, 878, 1114, 3283, 905, and 998 DEGs were found up- or down-regulated by drought in the samples of root at the tillering stage (TR), leaf at tillering stage (TL), root at panicle elongation stage (PR), leaf at panicle elongation (PL), young panicle sample at booting stage (BP), and leaf at booting stages (BL), respectively , response to endogenous stimulus , transcription regulation , metabolism such as lipid and carbohydrate metabolism , signal transcription , and cell wall and membrane component .In Figure http://www.tm4.org/mev.html). Results show that the DEGs can be classified into several groups according to the expression pattern: the first group was the up-regulated gene set in at least one tissue; the second group was down-regulated gene set in at least one tissue; and the third group indicates the genes were specifically induced or repressed in one unique tissue  under drought stress  identified as LEA proteins, four temperature-induced proteins , four dehydrin family proteins, and two protein phosphatase 2C family proteins, which were identified in a common induced gene set, involved in abiotic stress responsiveness. The others are related to amino acid and nucleotide metabolism, putative proteins, and proteins with unknown function.DEGs in all samples were further compared to screen the common up- or down-regulated genes by drought. We found a small set of genes commonly induced by drought stress in all tissues  was required. However, this did not hold true in others. Tissue-specific DEGs were screened out using the following strategy: the genes with more than five-fold change only in the leaf tissues at the three development stages under drought were identified as the leaf specifically up- or down-regulated gene set; the specific DEGs in roots and panicle were also identified as those in leaves. Stage-specific DEGs were identified as genes with more than five-fold change in two tissues at one development stage only. After removing the redundancies of the probes, a total of 110, 363, and 448 tissue-specific DEGs were identified in leaf, root, and panicle, respectively , SDR protein (Os07g0664600) involved in ABA biosynthesis , IAA26 . Seven pAREB1 regulates novel ABRE-dependent ABA signaling that enhances plant drought tolerance in vegetative tissues [A total of 76 genes were preferentially induced in the roots at the tillering and panicle elongation stages  related to membrane biogenesis and cell wall extension were confirmed to be exclusively down-regulated in young panicle , Histone H2A (Os03g0279200), Histone H3 (Os06g0160100), Histone H3 (Os05g0438700), and Histone H3 (Os01g0866200). These genes are involved in cell division and are reported to be regulated by drought and development [Five histone genes were detected to be highly down-regulated by drought exclusively in young panicle: 3 Os05g04700, and elopment . HoweverSeveral genes showing reciprocal expression patterns in two tissues at the same development stage under drought were also identified. After comparing the whole gene profiling between two tissues at the same development stage, several genes were detected to be reciprocally regulated in two tissues under drought. At the tillering stage, 5 and 6 genes were induced and repressed in leaves but repressed and induced in roots, respectively. At the panicle elongation and booting stages, 29 and 4 genes were identified to be reciprocally regulated by drought in two different tissues, respectively Table .GA2ox, Chitinase III, and Senescence-associated protein 15 (SAP15), were highly induced in leaf but repressed in root at both the tillering and panicle elongation stages. GA2ox was highly down-regulated in all roots but up-regulated in all leaves and panicle. One gene encoding the LEA protein (Os03g0322900) was only down-regulated in tillering leaf but up-regulated in all other tissues under drought. These results provide further evidence that the genes are regulated under environment stress in a tissue-specific manner. GA2ox has an important role in the regulation of stem elongation and leaf growth, and participates in the phase transition from vegetative to reproductive growth [GA2ox may result in a low level of GA in leaf under drought by feedback control and retardation of leaf growth, whereas the GA2ox is highly repressed by drought. Senescence-associated proteins are involved in leaf senescence in plant [SAP15 in root and leaf indicates that leaf growth was inhibited, whereas root growth was promoted by drought stress. The gene of Chitinase III confers disease resistance by degrading chitin, a component of fugal cell wall [Three genes, i.e., e growth . Inductiin plant . The recell wall . The bioOrgan-/tissue-specific gene regulation in response to abiotic stresses has been previously reported. The gene encoding glutamine synthetase in potato was found to have a differential response to drought and salt stresses in an organ-dependent manner ,43. A syAmong the 2384 known or annotated TF genes in the rice genome , 261 10, accountOsbZIP14, Os01g0867300) and HB (Os02g0649300) proteins were commonly induced in all tissues at the three development stages, indicating that these two TF genes might be involved in the universal regulation of rice response to drought. OsbZIP14 was also found to be induced by drought in all tissues during panicle and seed development [After comparing the expression patterns of all TF genes, different sets of TFs genes with unique expression patterns were identified. Two TF genes encoding bZIP  were found to be down-regulated in either at least one root tissue or one leaf tissue  by drought at booting stage. A total of 25 TF genes belonging to bHLH, GRAP-G2-like, MYB, NAC, and ZIM families were only differentially regulated (the same or different pattern in two tissues) by drought at the panicle elongation stage. There were 7 and 7 MYB and NAC genes induced by drought exclusively in leaf at panicle elongation stage, respectively  containing an ACGT core sequence  were used to search the upstream of the 55 genes with the control upstream sequences of 360 genes showing no significant change in expression pattern under drought. We found four elements over-represented in the commonly induced 55 genes compared with the control. A total of 72.7% of the genes contain 1-8 copies of the 4 ABREs, whereas only 3.6% of the control genes harbor 1-3 copies of the elements. Another ABRE motif, S000278 with ACGT core sequence, was also significantly detected to be over-presented in the 55 genes. Around 31.5% of the genes have at least one copy of S000278, whereas only 0.83% of the control genes have the sequence(s).cis-elements after further analysis; this special CGCG box is regulated by calmodulin and involved in the transcription regulation of multiple abiotic stresses responsiveness [physcomitrella ABA- and stress-induced gene set [All 1 kb sequences in the upstream of the 55 genes were submitted to a local installation of Weeder (version 1.3) and an extra mode search  was performed to look for the candidate motifs in a single strand. Four GC-rich sequences, namely, 6 bp of CCGCGC, 8 bp of CGCCGCGC, 10 bp of GCCGCGCGGC, and 12 bp of GCCGCGCCGCGC, were identified. The motif sequence logo is available in the supplementary material  and two donors, BR24 and Binam.Click here for fileTotal number of genes expressed in different samples under control and drought stressed conditions. Excel file containing the summary result of expressed gene number in different samples.Click here for fileHierarchical cluster analysis of six tissue types and all DEGs under drought stress. PPT file containing the result of the hierarchical cluster analysis.Click here for fileList of primers for the RT-PCR. Excel file containing all primer sequences used for the RT-PCR experiment.Click here for fileSemi-quantitative RT-PCR confirmation of microarray data. Description: A ppt file containing semi-quantitative RT-PCR confirmation of microarray data. The microarray data are shown on the left side, and the RT-PCR results are shown on the right side. A total of 21 genes were differentially regulated by drought at the tillering stage, panicle elongation stage, and booting stage. TLC and TLS indicate leaves under control and under stress, and TRC and TRS indicate root under control and stress at the tillering stage, respectively. PLC and PLS indicate leaves under control and stress, and PRC and PRS indicate root under control and stress at the panicle elongation stage, respectively. BLC and BLS indicate leaves under control and stress, and BPC and BPS indicate panicle under control and stress at the booting stage, respectively.Click here for fileComparative diagram of the tissue-specific DEGs under drought stress. A ppt file containing a comparative diagram of the total number of up-, down-regulated, and common regulated genes between leaves and root at the tillering stage , leaves and root at the panicle elongation stage , and leaves and panicle at the booting stage  under drought stress.Click here for fileVenn diagram of all tissue up- and down-regulated DEGs under drought stress. PPT file for the result of Venn Diagram of all tissue-specific DEGs.Click here for fileList of commonly induced genes by drought in all tissues at all development stages. Excel file containing the list of the commonly induced genes in all samplesClick here for fileList of commonly down-regulated genes by drought in all tissues at all development stages. Excel file containing the list of the commonly down-regulated genes in all samplesClick here for fileLeaf specific up-regulated genes under drought stress. Excel file containing all specific up-regulated genes by drought in leaf.Click here for fileLeaf specific down-regulated genes under drought stress. Excel file containing all specific down-regulated genes by drought in leaf.Click here for fileRoot-specific up-regulated genes under drought stress. Excel file containing all specific up-regulated genes by drought in roots.Click here for fileRoot-specific down-regulated genes under drought stress. Excel file containing all specific down-regulated genes by drought in rootsClick here for filePanicle-specific up-regulated genes under drought stress. Excel file containing all specific up-regulated genes by drought in panicleClick here for filePanicle-specific down-regulated genes under drought stress. Excel file containing all specific down-regulated genes by drought in panicleClick here for fileList of TF genes differentially regulated under drought stress. Excel file containing all transcription factor genes differentially regulated by drought stress in all samplesClick here for fileList of TF gene stages specifically regulated by drought stress. Excel file of the identified transcription factor genes specifically regulated by drought stressClick here for fileSequence Logo of the core sequences of the identified four motifs. A PPT file of the sequence logo of the core sequences of the identified motifsClick here for file


Caenorhabditis elegans uses gap junctions to provide direct cell-to-cell contact. The nematode gap junctions are formed by innexins ; a family of proteins that surprisingly share no primary sequence homology, but do share structural and functional similarity with connexins. The model organism C. elegans contains 25 innexin genes and innexins are found in virtually all cell types and tissues. Additionally, many innexins have dynamic expression patterns during development, and several innexins are essential genes in the nematode. C. elegans is a popular invertebrate model due to several features including a simple anatomy, a complete cell lineage, sequenced genome and an array of genetic resources. Thus, the worm has potential to offer valuable insights into the various functions of gap junction mediated intercellular communication.As in other multicellular organisms, the nematode  C. elegans were first described in 1996  in  Phelan, . FurtherC. elegans homotypic gap junctions have been suggested for the majority of innexins studied, namely INX-3, EAT-5, INX-6, INX-16 and INX-19 while UNC-7 and UNC-9 seem to be able to form both homotypic and heterotypic gap junctions. INX-14 and INX-22 are suggested to form heteromeric gap junctions, and these two innexins or perhaps only INX-14 is further suggested to form heterotypic gap junctions with INX-8 and/or INX-9. INX-19 is the only innexin for which a potential function as a hemichannel has been reported (see below for details).Gap junctions form when two hemichannels from adjacent cells line up to make a channel; in vertebrates the hemichannels are hexamers of connexin or pannexin proteins and in invertebrates the innexins are also believed to form hexameric channels , suggesting that all 25 innexins are true genes, not pseudogenes . In the adult worm, the INX-3 protein can only be detected in the posterior pharynx. Overexpression of inx-3 causes terminal defects during embryogenesis similar to the ones observed in the inx-3 mutant, so obviously this innexin is crucial for this developmental process, but the exact function remains unknown , non-parasitic, transparent nematode with a fairly simple anatomy and the adult animal has only 959 somatic cells. It is easily cultured in the laboratory on agar plates seeded with E. coil bacteria and it grows from embryo to fertile adult in approximately 3 days at 20° C. The worm goes through four larval stages, L1–L4, and then reaches adulthood. Limitations in food or other stress factors at the L1–L2 stage causes the larvae to enter dauer stage, a stress-resistant, and long-lived diapause stage. When food becomes available, the dauer will resume development. The total lifespan of C. elegans is around 3 weeks.C. elegans by feeding worms E. coil expressing dsRNA corresponding to the gene of interest. 40% of C. elegans genes have human orthologs (greater than 7000 genes).The animal is hermaphroditic and self-fertilizing, as it produces both oocytes and sperm. Hermaphrodites have two X chromosomes, while males have a single X chromosome. Male and hermaphrodite crosses can be done for genetic studies. Adult hermaphrodites, when self-fertilizing, will produce around 300 progeny. Mutant animals can be obtained by chemical mutagenesis and strains can be stored frozen for extended periods of time (decades). Many mutants can be maintained as self-fertilizing hermaphrodites on plates. Transgenic animals, for example animals expressing a GFP fusion protein, can be generated by microinjection or bombardment of exogenous DNA. RNA-mediated interference (RNAi) is readily obtained in C. elegans feeds by pumping bacteria into the pharynx, a tube-like muscular pump with its own nervous system. Bacteria trapped in the pharynx are broken up by muscle contractions and passed into the intestine, the digestive organ extending almost the entire length of the animal. The nervous system of the worm is comprised of 302 neurons and the complete wiring is known. Most neurons are located in the head of the worm, and cells of the nervous system are organized into ganglia in the head and tail. Body wall muscles run along the whole length of the animal in four quadrants and smaller muscles are located in the pharynx and around the intestine and the vulva. The reproductive system in the hermaphrodite consists of two gonadal arms joined by a shared uterus. Germ cells in the distal part of the gonad are undifferentiated and mature as they move proximally. The mature oocytes are ovulated into the sperm-containing spermatheca, where they are fertilized before moving into the uterus. Figure adapted from www.wormatlas.org.eat-5 mutants were named based on their abnormal eating; they display unsynchronized muscle contractions in the pharynx, the organ responsible for capturing bacterial food, grinding it up and transporting it to the intestine . Microinjection of low molecular weight fluorescent dyes indicated that the INX-6 channel is more permeable than connexin43, as the INX-6 channels allowed passage of 3 kDa and even some 10 kDa tracers. Curiously, INX-6 formed gap junctions when expressed in the insect cell line Sf9 but did not form gap junctions in mammalian HeLa cells, which could reflect the need of a modifier or modification for INX-6 to form actual channels  suggest that INX-16 forms homotypic channels, since the protein is absent from cell-cell contacts between inx-16 expressing and non-expressing intestinal cells  they mature and the oocytes will move into the sperm-containing spermatheca, where they are fertilized see Box . Subsequfog-2(q71) background, which generates XX females and XO males] , which acts as a paracrine hormone and induces oocyte maturation and sheath contraction. The extracellular MSP gradient in the gonad of hermaphrodites is not believed to be high enough to reach the loop region of the gonad where stimulation of oocyte maturation takes place, and the sheath cells are hypothesized to communicate the presence of the MSP signal throughout the whole germline since the sheath cells are coupled to one another and to the germline via gap junctions. The two screens identifying inx-14 and inx-22 as negative regulators of oocyte maturation were both RNAi screens and would not have picked up any innexins with redundant functions  , showing that INX-19 localized to plaques at cell-cell contacts when both cells were expressing this innexin, suggesting the formation of a homotypic gap junction  is expressed in a subset of neurons in the head and tail and was identified in a genetic screen for mutants lacking asymmetrical AWC olfactory neurons. The pair of AWC neurons are morphologically symmetric, but they differ in their ability to sense different chemicals due to different genes being expressed in either the left or right neuron (Chuang et al., ON and one AWCOFF neuron are always generated, where the ON and OFF denotes the expression of the reporter str-2::gfp. Removal of inx-19 caused two AWCOFF neurons, whereas overexpression of inx-19 generated two AWCON neurons, clearly showing the importance of this innexin in establishing the asymmetry of the AWC neurons. By expressing vertebrate Ca2+-buffer proteins within the INX-19 network, the AWC asymmetry was disrupted, thereby indicating that Ca2+ signaling is involved in establishing the left-right asymmetry in C. elegans AWC olfactory neurons via an INX-19 transient gap junctional network (Schumacher et al., In wild type animals, one AWCC. elegans has 25 innexins and, so far, in-depth functional analysis has been done for less than half of the genes including inx-3, inx-6, eat-5, inx-16, inx-14, inx-22, inx-19, unc-7, and unc-9. All these innexins seem to act as genuine gap junctions, as evidenced by a variety of techniques; electron microscopy, Xenopus oocyte coupling, expression of innexins in COS cells and Sf9 cells, fluorescent dye transfer assays, studies of GFP fusion-proteins, voltage clamp and RNAi. One of the most interesting results to come from these studies was the identification of a modifier of gap junctions formed by UNC-9, namely the stomatin-like protein UNC-1 (Chen et al., C. elegans innexins. Another important finding is that channels formed by C. elegans INX-6 show a more permeable gap junction with a larger channel diameter than has been reported for connexins (Oshima et al., C. elegans have emerged from these studies including roles in movement, fertilization, oocyte maturation, cell identity and innate immunity. In other words, innexins are involved in almost all aspects of the development of this organism.C. elegans these approaches have been used in various cell types. When carboxyfluorescein was injected into muscle cells of dissected pharynxes, it spread to the remainder of pharyngeal muscles in wild type worms, but not in eat-5 mutant animals (Starich, C. elegans and here the dye transferred throughout the intestine (Peters et al., When studying gap junctions, dye transfer assays and specific inhibitors are often used, and in Starich, . AnotherC. elegans is a small animal model with advantages for studying cell-cell communication, and this nematode has the potential to contribute a great deal to our understanding of how gap junctions function.Although the vertebrate connexins and invertebrate innexins are completely unrelated by sequence, they are both the molecular building blocks of gap junctions and thus enable intercellular communication. Much is still unknown for innexins—Are they post-translationally modified? Can they form functional hemichannels? Do any of them have channel-independent functions? Perhaps the biggest question still to be answered is, what small molecules are transported through the innexin gap junctions? The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.