Our results challenge conventional assumptions and reported findings that human impacts to ecosystems are cumulative and lead only to long-term trajectories of environmental decline. In contrast, recovery periods reveal that human societies have interacted sustainably with coral reef environments over long time periods, and that degraded ecosystems may still retain the adaptive capacity and resilience to recover from human impacts.Coral reef ecosystems are declining worldwide, yet regional differences in the trajectories, timing and extent of degradation highlight the need for in-depth regional case studies to understand the factors that contribute to either ecosystem sustainability or decline. We reconstructed social-ecological interactions in Hawaiian coral reef environments over 700 years using detailed datasets on ecological conditions, proximate anthropogenic stressor regimes and social change. Here we report previously undetected recovery periods in Hawaiian coral reefs, including a historical recovery in the MHI (∼AD 1400–1820) and an ongoing recovery in the NWHI (∼AD 1950–2009+). These recovery periods appear to be attributed to a complex set of changes in underlying social systems, which served to release reefs from direct anthropogenic stressor regimes. Recovery at the ecosystem level is associated with reductions in stressors over long time periods (decades+) and large spatial scales (>10 Coral reefs are among the most diverse and productive ecosystems worldwide, but the abundance of key species and habitat builders have declined globally due to human activities Restoring ecological resilience requires understanding long-term trends (decades to centuries) in resource and ecosystem conditions and characterizing the complex ways that societies have mediated environmental outcomes in the past 3 km2]), which limited direct human stressors of reef systems to sustainable levels. Reductions in proximate human stressors were mediated indirectly by a complex set of historical events and consequent changes in social systems, which altered social-ecological relationships in reef environments.Using coral reefs and island societies as a model social-ecological system th century We reconstructed ecological changes at the guild level, relying on a diversity of data types to assess changes in the degradation and depletion of coral reef biota through an intensive review and assessment of archaeological deposits, ethnohistoric and anecdotal descriptions and modern ecological and fishery data. This type of historical analysis is limited in precision but assessing change through multiple data types provides a valid method for reconstructing long-term ecological trends and characterizing the social factors that have shaped ecosystem conditions Coral reef ecosystem conditions were reconstructed using a modified version of a method developed for assessing long-term ecological change in marine environments Data were extracted directly or derived from archaeological studies and midden analyses , ethnographic and archival anecdotal accounts (N = 990), contemporary ecological studies (N = 228), and annually published fisheries datasets (N = 55) . ArchaeoThe status of ecological guilds was determined through analysis and quantitative scoring of data sources . QuantitTransitions between ordinal EcoState scores in individual guilds were scored as to whether the changes were gradual or sudden based on the evidence available from different data types. For example, the transition from subsistence use to mass harvesting of a marine resource for a newly developed export market constituted a sudden transition (e.g. from EcoState 2→3), whereas if evidence revealed a gradual decrease in the abundance of an organism then the transition was scored as a gradual shift (e.g. from EcoState 1→1.25→1.5→1.75→2). To determine the overall trajectory of the entire coral reef ecosystem, EcoState scores for each guild were averaged. Averages were not weighted, which assumes that each guild is equally important in maintaining an intact coral reef. Ordinal EcoState scores  were conProximate human stressors to coral reef ecosystems were quantified for each guild through time, and were classified into five major types of disturbance: 1. Overexploitation; 2. Invasive Species; 3. Land-Based Pollution, 4. Disease; and, 5. Climate Change. Proximate stressors were quantified on an ordinal scale that included small (1), medium (2) or large (4), and the magnitude of proximate stressors was determined based on a determination of severity of the effect through reviewing all the available evidence. For example, overexploitation by subsistence or artisanal fishing was determined to be small (1), whereas evidence of large-scale commercial or industrial harvesting was classified as large (4). To quantify proximate stressors at the ecosystem level, individual stressors were summed for each guild. Cumulative human impacts were then represented graphically on an intensity scale as a proportion of the total stressor load by region (MHI & NWHI). Underlying social factors were determined by reconstructing detailed timelines of social change in demography, economic systems, technologies and human institutions related to coral reef environments. Changes in these social systems were described in terms of impacts to each ecological guild, and linkages between social change, proximate human stressors, and ecological conditions were assessed based on the available evidence from multiple sources.Our reconstruction reveals that trajectories of change in ecological condition differed among coral reef guilds and between regions of the Hawaiian archipelago . GeneralVoyaging Polynesians arrived in the Hawaiian archipelago sometime around AD 1250 To analyse archaeological data, we relied on well-established models derived from foraging theory to infer ecological impact on the basis of loss in abundance, size or diversity of taxa found in midden deposits The archaeological record in Hawai‘i exhibits considerable variation in patterns of marine resource exploitation The considerable intra-site variability led us to examine a few well-described midden assemblages as representative sites to assess temporal patterns in subsistence strategies and estimate the ecological impact of human exploitation on reef ecosystems in prehistory (AD 1250–1778). We purposefully selected sites capable of supporting both hunter-gatherer activities and agricultural activities (‘mixed-use’ sites), which we felt were the most appropriate as representative sites for assessing regional-level changes in subsistence patterns. Considerable care must be taken in extrapolating ecological conditions from patterns of recovered faunal remains in archaeological sites Our detailed analysis of faunal remains from these representative sites reveals a pattern of decline in reef fish relative to domesticated mammals as a primary source of meat-derived protein in Hawaiian prehistory . This anBy the late prehistoric period (> AD 1400), however, reef-derived protein sources are less prevalent than domesticates, suggesting a shift in the basic modes of subsistence and exploitation patterns in prehistoric Hawaiian societies . The treIn sum, intra-site trends in faunal remains suggest partial resource recovery as a result of decreased and differential exploitation of reef ecosystems from AD 1400–1800. Competing, but we suggest less likely, hypotheses that may serve to explain these trends include environmental changes affecting the availability of certain species, prey release due to reduction of higher trophic level predators, sampling artefacts, or changes in preferences or cultural traditions that influenced the collection of reef species. We conservatively estimate this recovery as not exceeding 20% for free-living guilds , which tWe also conducted a comprehensive review of observations made by explorers, traders and merchants, whaling and sealing crew members and captains, missionaries and Native Hawaiians about Hawaiian coral reef environments in the early historic period after western contact (AD 1778–1850+). Such observational data are often limited in precision but nonetheless can be used to derive valid information about the condition of marine ecosystems and social relationships with these environments Carangidae]), and in the late 1780s Nathaniel Portlock purchased “snappers, rock-cod, [reef piscivores]” from Ni‘ihau. In the mid-1780s, Colnett noted a high abundance of both fish and sea turtle at Ni‘ihau and described that Hawaiians from the southwest shore of O‘ahu had only “great quantitys” [sic] of fish to sell. Members of Vancouver's expedition in the early 1790s also described the large quantity of salted fish available at Ni‘ihau, and reef fish were so plentiful that they were traded to the people of Kaua‘i for cloth and mats. Quimper purchased “excellent snails” [intertidal/reef invertebrates] at both Kealekekua and at Waimea on Kaua‘i. Early visitors also described high abundances of predators in Hawaiian reef environments  declined precipitously in the 1970s–80s due in part to concerns about ciguatera poisoning, and these species are now abundant in the NWHI Following WWII, commercial fishing operations continued but the domestic fleet remained fairly small over the past century (<12 vessels) Coral reefs remained a focus for resource extraction throughout Hawaiian prehistory, but the development of large-scale agricultural and aquaculture complexes and increases in animal husbandry prevented societies from relying exclusively on reef environments for food resources. Our research suggests that reef recovery in the MHI during the late prehistoric period was probably attributed to shifts in the basic patterns of subsistence . These sThe pattern observed in Hawaiian prehistory is similar to other island systems where the success of human colonizers occurred at the expense of vulnerable taxa Subsistence exploitation in prehistory altered the structure of coral reef ecosystems through the removal of large predators and high-value prey, similar to changes observed in modern contexts After western contact, reef recovery in the MHI is attributed to extensive indigenous depopulation due to disease epidemics and changes in labor and the modes of production , Table 1In the NWHI, reef recovery is associated with 100+ years of environmental protections, human depopulation, ecological restoration efforts and decreases in the extractive capacity, spatial extent, and range of species targeted by commercial operations , Table 13 km2). Though the underlying social factors responsible for recovery differ among regions  of proximate stressors are reduced over long time periods (decades+) and large spatial scales  are displayed on the right side of the graph, and observations are plotted by each year for each guild on the horizontal axis. Archaeological data are displayed as one observation per earliest reliable date of the excavated site, but most sites spanned longer time series than are graphically indicated Click here for additional data file.Figure S2Changes in ecological conditions through time for free-living guilds  for the Main Hawaiian Islands (A–D) and the Northwestern Hawaiian Islands (E–H). The vertical axis represents ecological condition as a percentage of depletion-degradation, with 0% = pristine and 100% = globally extinct. Time is represented on the horizontal axis (AD 1250–2009); 100-year increments are used in the prehistoric period (AD 1250–1778) and 20-year increments are used for the historic and contemporary period (AD 1778–2009).(TIFF)Click here for additional data file.Figure S3Changes in ecological conditions through time for architectural guilds  for the Main Hawaiian Islands (A–C) and the Northwestern Hawaiian Islands (D–F). The vertical axis represents ecological condition as a percentage of depletion-degradation, with 0% = pristine and 100% = globally extinct. Time is represented on the horizontal axis (AD 1250–2009); 100-year increments are used in the prehistoric period (AD 1250–1778) and 20-year increments are used for the historic and contemporary period (AD 1778–2009).(TIFF)Click here for additional data file.Figure S4Trajectories of change in free-living  and architectural/sedentary guilds  from AD 1250–2009 for the Main Hawaiian Islands (MHI)  and the Northwestern Hawaiian Islands (NWHI) (dark & light blue lines). Reef recovery in the MHI (∼AD 1450–1800) and the NWHI (∼AD 1950–2009) was driven primarily by free-living guilds.(TIFF)Click here for additional data file.Figure S5Comparison of Hawaiian coral reef ecosystem trajectories with global estimates of reef conditions. Trajectories include the Main Hawaiian Islands  and the Northwestern Hawaiian Islands . Periods of reef recovery in the MHI (AD 1400–1820) and the NWHI (AD 1950–2009) are indicated where the trend line is yellow. Current ecosystem conditions for the Hawaiian Islands are compared with global assessments of coral reefs reported by Pandolfi et al. (2003), which shows that regions in the Hawaiian archipelago  occupy the distal ends of the global spectrum of observed conditions in coral reef ecosystems on an axis of depletion-degradation. Time is represented on the horizontal axis (AD 1250–2009); on the horizontal axis 100-year increments are used in the prehistoric period (AD 1250–1778 AD) and 20-year increments are used for the historic and contemporary period (AD 1778–2009).(TIFF)Click here for additional data file.Figure S6Human population in Hawai‘i, AD 1250–2010. A demographic model developed by Dye and Komori (1992) (as reviewed by Kirch 2007b), was used for estimating pre-censal population, which remains poorly understood and the subject of some controversy (Kirch 2007b). High and low bounds (grey lines) and an estimated mean (black line) are presented for this period (AD 1250–1832). Census data were used for the period 1853–2010  (black line).(TIFF)Click here for additional data file.Table S1Descriptions and examples of biota comprising of coral reef guilds.(DOCX)Click here for additional data file.Table S2Ecological states (‘EcoStates’) and criteria used to assess the condition of coral reef guilds for different data types on an ordinal scale. General criteria are presented as headings in each row of the second column. General criteria developed by Jackson et al. (2001) and Pandolfi et al.  were further refined to evaluate the level of impact by data type. These criteria were developed in recognition that the quantitative scoring of different studies and data types required a common methodology for evaluation, but that specific criteria were needed in order to evaluate each data type. For example, corals are rated by the total amount that have declined or are at risk, with 0–10% at risk being pristine, 11–29% abundant/common, 30–59% depleted/uncommon, 60–89% rare, 90–99% ecologically extinct, and 100% globally extinct. For states 2–5, more specific criteria used for each type of data  are described.(DOCX)Click here for additional data file.Table S3Summary of data types and number of records used to reconstruct ecological conditions through time. Archaeological records include published studies on marine fauna from archaeological sites See , below. (DOCX)Click here for additional data file.Table S4Archaeological sites in the MHI and studies that report marine fauna in midden deposits. Citation references are in Appendix A. NWHI sites (not listed in table) include sites on Nihoa and Mokumanamana (Necker) Islands . No midden remains have been discovered in other NWHI sites .(DOCX)Click here for additional data file.Table S5Summary of trends in faunal remains recovered from excavated middens in archaeological sites in the main Hawaiian Islands, sorted by date. Changes in ecological state (EcoState) are denoted as positive (up arrow), negative (down arrow), or not able to be determined (−) for specific time periods based on summary of major findings. Abbreviations for ecological guilds as follows: SC  =  small carnivores; SH  =  small herbivores; LH  =  large herbivores; LC  =  large carnivores. *indicates sites that have not been re-dated with modern methods; as such dates must be viewed with caution. Numbered citations are listed below the table.(DOCX)Click here for additional data file.Table S6Representative observations of the ecological condition of reef biota in the Main Hawaiian Islands during the prehistoric and early historic period. *exact dates for these observations are not described; estimates are derived from descriptions of authors or commentators.(DOCX)Click here for additional data file.Supporting Information S1Bibliography of sources.(DOCX)Click here for additional data file.Supporting Information S2Summary narratives of the primary data used to reconstruct ecological conditions and anthropogenic impacts are presented by guild. At the beginning of each guild subsection, tables summarize a timeline specific to the guild and include descriptions of major events, intensity of proximate stressors and the quantitative EcoState scores assigned to different time periods (Tables A–F). In the summary narratives that follow these tables, a chronological overview synthesizes primary data from multiple data sources and types. These syntheses by guild justify the quantitative scores determined for guild EcoStates and proximate stressor regimes. Ecological changes are reconstructed from multiple lines of evidence. Guild summary narratives are supplemented with additional data in the form of endnotes. In the narratives, terms are used to refer to specific periods in Hawaiian history; these include: 1) prehistoric: AD 1250–1778; 2) historic: AD 1778–1900; and, 3) modern: AD 1900+.(DOCX)Click here for additional data file.


Sorting of cargoes in endosomes occurs through their selective enrichment into sorting platforms, where transport intermediates are generated. The WASH complex, which directly binds to lipids, activates the Arp2/3 complex and hence actin polymerization onto such sorting platforms. Here, we analyzed the role of actin polymerization in the physiology of endosomal domains containing WASH using quantitative image analysis. Actin depolymerization is known to enlarge endosomes. Using a novel colocalization method that is insensitive to the heterogeneity of size and shape of endosomes, we further show that preventing the generation of branched actin networks induces endosomal accumulation of the WASH complex. Moreover, we found that actin depolymerization induces a dramatic decrease in the recovery of endosomal WASH after photobleaching. This result suggests a built-in turnover, where the actin network, i.e. the product of the WASH complex, contributes to the dynamic exchange of the WASH complex by promoting its detachment from endosomes. Our experiments also provide evidence for a role of actin polymerization in the lateral compartmentalization of endosomes: several WASH domains exist at the surface of enlarged endosomes, however, the WASH domains coalesce upon actin depolymerization or Arp2/3 depletion. Branched actin networks are thus involved in the regulation of the size of WASH domains. The potential role of this regulation in membrane scission are discussed. The Arp2/3 complex is a major actin nucleator that generates branched actin networks at different cellular locations Actin polymerization at the surface of endosomes was previously involved in the regulation of vesicle fusion The identity of the endosomal transport intermediate, containing specific cargoes, is conferred by its content in Rab GTPases that defines its target destination Here we investigated the role of actin polymerization at the surface of endosomes by monitoring the WASH marker, which defines an endosomal domain. Using quantitative image analysis, we identified two specific feedback regulations of the WASH domain by the branched actin network it nucleates: actin filaments control the lateral organization of WASH domains and the dynamic exchange of WASH molecules between the endosomal domain and the cytosolic pool. The potential role of these feedback regulations in the mechanism of endosomal scission is discussed.To analyze the role of actin dynamics in the physiology of endosomal WASH domains, 3T3 cells were loaded with fluorescent transferrin (Tf) to label sorting and recycling endosomes, then treated with actin depolymerizing drugs in the presence of Tf. We used Latrunculin A (LatA), which sequesters monomeric actin and prevents it from polymerizing To confirm this result more quantitatively, we needed an accurate statistical analysis to measure precisely the WASH signal on each endosome. This obviously required the accumulation of data over thousands of endosomes, due to their high heterogeneity in size and shape. We thus additionally treated the cells with nocodazole, which depolymerizes microtubules and therefore prevents endosome clustering around the nucleus. In these conditions, we were able to resolve almost all endosomes. In these conditions as well, the WASH signal appeared brighter in LatA-treated cells than in control cells . We veriUpon LatA treatment, endosomes appeared larger, consistently with previous observations 3 (2 test). Moreover, this analysis showed that in the control situation, WASH domain intensity increases with the volume of the endosome  large endosomes have larger WASH domains than smaller ones, but with a similar surface density of WASH, (ii) in contrast, actin polymerization negatively regulates the surface density of WASH in its domain.To evaluate the dynamics of WASH when actin polymerization is impaired or not, we used Fluorescence Recovery After Photobleaching (FRAP) of GFP-WASH. WASH domains of similar size were bleached in control cells and in LatA treated cells. The bleached zone was large enough to encompass the whole endosome ensuring that fluorescence recovery was due to recruitment of cytosolic WASH and not to lateral diffusion . RecoverHowever, LatA depolymerizes all actin networks, whether the Arp2/3 complex had nucleated them or not. We thus depleted the Arp2/3 complex to specifically impair the generation of branched actin networks. Transfection of 3T3 cells with siRNAs targeting p34Arc induced a strong decrease in the protein levels of p34Arc and p16Arc, suggesting depletion of the entire complex . Arp2/3 We often observed elongated GFP-WASH-positive structures on large endosomes upon Arp2/3 depletion . Such st2 for endosomes larger than 1 µm3. When LatA is applied, the increase in the number of WASH domains as a function of endosome volume is strongly diminished. For the largest endosomes, it culminates at 2 WASH domains per endosome, compared to 5 in the control situation. Concomitantly, the apparent surface that each WASH domain occupies increases steadily to 2.5 µm2 for the largest endosomes. All the above observations were statistically significant  large endosomes have more WASH domains than smaller ones; (ii) actin polymerization constrains the surface area of WASH domains below an upper limit.To directly demonstrate that actin depolymerization induces coalescence of WASH domains, we sought to image the process after LatA addition. To alleviate the problem of out of plane WASH domains on enlarged endosomes, we acquired 12 z-planes by spinning disk confocal microscopy every 30 seconds, and performed a maximum intensity z-projection of the planes . This exWe initially reported that WASH belongs to a stable multiprotein complex We next examined whether coalescence upon actin depolymerization is a general behavior of endosomal domains or a specificity of WASH domains. The Vps35 subunit of the so-called ‘cargo selective subcomplex’ of the retromer was recently shown to bind directly to the FAM21 subunit of the WASH complex and to be required for the endosomal recruitment of the WASH complex Using LatA mediated actin depolymerization, we have observed two effects on the Arp2/3 activator WASH at the surface of endosomes. The first one is an increased WASH staining on endosomes and the second one is the coalescence of WASH domains at the surface of large endosomes. We are confident that these two effects are not due to artifactual antibody staining, as they were similarly observed using a GFP-WASH fusion protein. These effects were best seen when endosomes were scattered using microtubule depolymerization. However, the increased WASH staining and the crescent shaped WASH domain were also observed without nocodazole, suggesting that these alterations of WASH distribution solely rely on the actin cytoskeleton. Indeed, Arp2/3 depletion produced the same phenotype, thus ruling out non specific effects of LatA treatment. We attempted to confirm the endosomal WASH enrichment that we have observed upon actin depolymerization using established procedures for membrane cytosol biochemical fractionation assays To accurately quantify these effects on WASH distribution upon LatA treatment, we had to develop automated image analysis techniques to assign each WASH domain to its correct endosome in 3D. Specifically, our method overcomes the acute problem of the heterogeneity of size and shapes of endosomes. This method helped us to accumulate sufficient data to prove that the two effects on WASH distribution we report here are indeed general effects independent of the variable sizes and shapes of endosomes. We anticipate that these programs, available on request, will be useful for future endosomal studies, for instance to quantify protein recruitment onto endosomes and endosomal protein contents in vivo.Arp2/3 depletion phenocopies LatA treatment on WASH distribution, thus suggesting that WASH is controlled by the branched actin networks it generates through feedback mechanisms. Indeed, both treatments induce an accumulation of WASH on endosomes. Moreover, FRAP experiments on GFP-WASH have revealed that LatA treatment ‘freezes’ the dynamic exchange of WASH between cytosolic and endosomal pools. The lack of WASH recovery after photobleaching indicates that cytosolic WASH is less recruited to endosomes upon LatA treatment. There are two possible interpretations to explain this effect. Either the recruitment of WASH from the cytosol requires actin – however, it is difficult to envision in this case why the endosomal pool of WASH increases upon LatA treatment – or, it is the detachment of WASH from endosomes that requires actin. We favor this hypothesis, which is consistent with the increased endosomal pool of WASH upon actin depolymerization. This hypothesis is less intuitive as to why there is less recovery of WASH after photobleaching. It is likely that defective recovery of GFP-WASH in this hypothesis is due to the constant occupancy of endosomal binding sites by bleached WASH, which do not detach to return to the cytosol.Such a feedback mechanism of actin networks on other Arp2/3 activators has been observed in different systems. Actin depolymerization increases the membrane pool of WAVE complex and freezes its dynamics in neutrophils We have also observed that branched actin networks control the lateral distribution of WASH domains at the surface of endosomes, since actin depolymerization induces coalescence of the WASH domains. This spontaneous coalescence of WASH domains is reminiscent of the behavior of lipidic domains at the surface of artificial vesicles Our hypothesis to explain WASH coalescence is centered on a platform containing specific lipids clustered by the WASH complex. The WASH platform would have an intrinsic coherence that is independent of actin, and a long-range organization that depends on the branched actin network it is associated with. The short-range coherence accounts for the coalescence of two platforms when they meet at the surface of endosomes and for the fact that WASH molecules remain in clusters without actin. Without such a short-range coherence, we would expect WASH molecules diffusing all over the surface of endosomes, which we did not observe with or without actin. This short-range coherence might arise from WASH induced lipid repartitioning and domain formation, as previously described for some viral or bacterial lipid binding proteins The WASH complex might promote the scission of transport intermediates by several mechanisms, which collaborate. First, it recruits dynamin, directly or indirectly through the cortactin-positive branched actin network it generates Flip-In 3T3 cells (Invitrogen) and 3T3 GFP-WASH stable cells were handled and transfected as previously described Full length human Vps35 was amplified by PCR using a template provided by L. Johannes  and cloned into a modified pCDNA5/FRT/V5-His (Invitrogen) vector, in which the CMV promoter was replaced by a EF1α/HTLV chimera promoter and which tags the ORF by a N-terminal eGFP Whole cell lysates were prepared by resuspending cells directly in SDS-PAGE loading buffer. Viscosity of samples after boiling was reduced using Benzonase (Sigma). SDS-PAGE was performed using NuPAGE 4–12% Bis-Tris gels (Invitrogen) transferred onto nitrocellulose membranes according to the manufacturer's instructions. Western blots were revealed using HRP coupled antibodies, Supersignal kit (Pierce) and a Fuji LAS-3000 (Fujifilm).For standard imaging of fixed samples, 3T3 cells were plated for 2 h onto glass coverslips coated with 50 µg/ml fibronectin (Sigma) before each experiment, fixed in PBS-3% paraformaldehyde, permeabilized in PBS containing 0.05% saponin, then processed for indirect immunofluorescence using standard techniques. Samples were then mounted in ProLong® Gold antifade reagent (Invitrogen).To preserve the structure of enlarged endosomes, cells co-expressing mCherry-Rab5Q79L and GFP-WASH or GFP-Vps35 were plated for 2 h onto fibronectin-coated surfaces , fixed in PBS-3% paraformaldehyde for 10 min at 37°C, processed for immunofluorescence as described above, then washed and imaged in PBS. For time-lapse microscopy, cells were plated for 2 h onto fibronectin-coated glass (Iwaki) or plastic  plates and imaging was performed in DMEM medium supplemented with 10 mM Hepes (Invitrogen).2 camera .Epifluorescence microscopy was performed as previously described http://rsb.ingo.nih.gov/ij/).Confocal sections were acquired using two custom spinning disk setups based on TE2000-U or TI-Eclipse inverted microscopes , equipped with temperature control chambers , 100× oil immersion objectives (NA 1.45 or 1.49), CSU22 or CSU-X1 spinning disk heads , CoolSnap HQ2 or Evolve EM-CCD cameras , and operated by Metamorph 7.1.4 or 7.6 , respectively. The TE2000-U was equipped with a piezoelectric translator mounted on the objective to control the position of the confocal plane . For dual-color live imaging, channels were acquired sequentially and, for each time point, several planes were acquired and projected to compensate the exit of endosomes from the confocal plane. Analyses of images and movies were performed using Metamorph and ImageJ softwares . 3D reconstructions were performed using Amira software  on deconvolved images. Movies were edited with Adobe Premiere software.For preliminary quantification , confocaFor automated analysis on scattered endosomes  and S3, To assign each WASH domain to its endosome, we developed a custom-made code in MATLAB . First, a common pixel yellow stack is generated from the segmented images of both green (WASH) and red (Tf) channels. This stack is then segmented and each yellow region is assigned to its red and green object, from which the association between the red and the green regions is deduced. Two geometrical methods were used to assign each Yellow object to its Green or Red object. First, a ‘Minimum Distance’ method finds the closest Green and Red objects for each Yellow object, based on the 3D-distance between geometrical centers. Second, a ‘Bounding Box’ method assigns a Yellow object to a Green (or Red) object if the fluorescence centroid of the Yellow object is contained within the minimum bounding box containing the Green (or Red) object. We excluded all the objects for which both methods gave different results . This MATLAB code is available on request.2 (XY-spacing×Z-spacing).For cells expressing mCherry-Rab5Q79L , image aData were plotted using Matlab and statistical tests were performed using Matlab or Sigmastat (Systat software). Endosome volume distributions were compared with the Chi-2 test while pooling volume bins superior to 1.4 to have more than 5 endosomes per bin. The preliminary analysis of the influence of actin depolymerization on the average WASH domain intensity  was analFRAP experiments were performed on the second spinning disk confocal microscope setup, using a 100× NA 1.40 PlanAPO objective and a FRAP module equipped with 491 and 642 nm lasers . Circular regions centered on WASH domains (10 px in diameter) were selected (∼5–10 regions/cell), and bleached for 50 ms using the 491 nm laser. Recovery was monitored on a single plane for 3 min , acquiring GFP-WASH and Alexa647-Tf channels sequentially using a µs-AOTF switch. FRAP analysis was performed with ImageJ using custom-made macros unless stated otherwise. First, the background was homogenously subtracted and the global photobleaching was corrected over the entire image. Then, WASH domains were manually tracked using the ‘MTrackJ’ ImageJ plugin developed by Eric Meijering. Finally, the fluorescence of WASH domains was integrated at each time point in a circular region (6 px in diameter) centered on the fluorescence centroid. Each WASH domain trajectory was examined and compared to the trajectory of its counterpart in the Tf channel. Aberrant trajectories and recoveries were discarded.Figure S1LatA treatment induces close to complete depolymerization of branched actin networks associated with WASH-positive endosomes. (A) 3T3 cells were treated as in (TIF)Click here for additional data file.Figure S2CytoD treatment increases endosomal WASH similarly to LatA. (A) 3T3 cells were loaded with fluorescent Tf until equilibrium, then treated with 10 µM nocodazole in the continuous presence of Tf for 1 h, then treated with 1 µM CytoD or carrier in the presence of nocodazole and Tf for 30 min. Cells were then processed for immunofluorescence and observed as in (TIF)Click here for additional data file.Figure S3Actin depolymerization increases the amount of GFP-WASH associated with endosomes. (A) Stable 3T3 cells expressing GFP or GFP-WASH were analyzed by Western Blot using indicated antibodies. GFP-WASH overexpression is limited. GFP-WASH replaces endogenous WASH in the stable cell line. (B) Cells expressing GFP-WASH were treated as in (TIF)Click here for additional data file.Figure S4FAM21 colocalizes with WASH, whether or not actin is depolymerized. (A) Stable 3T3 cells expressing GFP-WASH were treated as in (TIF)Click here for additional data file.Movie S1FRAP analysis of GFP-WASH dynamics. Several WASH domains were bleached in a nocodazole-treated cell. The bleached regions are indicated by circles in the 6 frames before bleaching. The insets show WASH domains (green) and the Tf-positive endosomes (red). Frame rate: 20 images per second. Scale bar: 10 µm.(MOV)Click here for additional data file.Movie S2Actin depolymerization inhibits WASH recovery on endosomes after photobleaching. This movie corresponds to (MOV)Click here for additional data file.Movie S3Depletion of the Arp2/3 complex induces the formation of enlarged endosomes harboring a crescent-shaped WASH domain. (MOV)Click here for additional data file.Movie S4Actin depolymerization by LatrunculinA induces the formation of enlarged endosomes harboring a crescent-shaped WASH domain. 3T3 cells stably expressing GFP-WASH were treated as in (MOV)Click here for additional data file.Movie S53D reconstructions of WASH domains on Rab5Q79L-enlarged endosomes in the presence or not of LatrunculinA. (MOV)Click here for additional data file.Movie S6Direct observation of WASH domain coalescence upon actin depolymerization. Two examples are shown. (MOV)Click here for additional data file.


Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that causes death within a mean of 2–3 years from symptom onset. There is no diagnostic test and the delay from symptom onset to diagnosis averages 12 months. The identification of prognostic and diagnostic biomarkers in ALS would facilitate earlier diagnosis and faster monitoring of treatments. Gene expression profiling (GEP) can help to identify these markers as well as therapeutic targets in neurological diseases. One source of genetic material for GEP in ALS is peripheral blood, which is routinely accessed from patients. However, a high proportion of globin mRNA in blood can mask important genetic information. A number of methods allow safe collection, storage and transport of blood as well as RNA stabilisation, including the PAXGENE and TEMPUS systems for the collection of whole blood and LEUKOLOCK which enriches for the leukocyte population. Here we compared these three systems and assess their suitability for GEP in ALS. We collected blood from 8 sporadic ALS patients and 7 controls. PAXGENE and TEMPUS RNA extracted samples additionally underwent globin depletion using GlobinClear. RNA was amplified and hybridised onto Affymetrix U133 Plus 2.0 arrays. Lists of genes differentially regulated in ALS patients and controls were created for each method using the R package PUMA, and RT-PCR validation was carried out on selected genes. TEMPUS/GlobinClear, and LEUKOLOCK produced high quality RNA with sufficient yield, and consistent array expression profiles. PAXGENE/GlobinClear yield and quality were lower. Globin depletion for PAXGENE and TEMPUS uncovered the presence of over 60% more transcripts than when samples were not depleted. TEMPUS/GlobinClear and LEUKOLOCK gene lists respectively contained 3619 and 3047 genes differentially expressed between patients and controls. Real-time PCR validation revealed similar reliability between these two methods and gene ontology analyses revealed similar pathways differentially regulated in disease compared to controls. Amyotrophic lateral sclerosis (ALS) is a devastating and fatal disease that preferentially affects the motor system. Clinically, ALS manifests as progressive weakness of voluntary muscles and patients survive on average for 2–3 years after onset of symptoms. The mechanisms that cause neurodegeneration in ALS are incompletely understood, and are considered to operate through a number of molecular and genetic pathways including glutamate toxicity, oxidative stress, the formation of protein aggregates and defects in axonal transport Gene expression profiling (GEP) is a powerful tool to help identify potential diagnostic and therapeutic targets in neurological diseases Conventional methods for reducing the relative amounts of globin mRNA in blood samples by density gradient centrifugation and extraction of the white blood cell population have been shown to be effective in reducing globin interference. However, the long duration of experimental handling during the extraction process leads to RNA degradation and possible unintended gene induction that might affect the validity of the disease related changes in gene expression In order to discover the best method of RNA isolation from blood to carry out GEP in a neurological disorder such as ALS, we compared these three commercially available systems for RNA extraction from blood. PAXGENE (PAX) and TEMPUS (TEM), which are column based methods leading to RNA extracted from whole blood, and LEUKOLOCK (LL), a method for isolating white blood cells for subsequent RNA extraction. We used a cohort of 8 sporadic ALS patients and 7, age-matched controls. RNA from PAX and TEM extractions also underwent a step to deplete globin using GlobinClear . Therefore, RNA from 5 experimental conditions  was hybridized onto U133 Plus 2.0 Human whole genome arrays (Affymetrix) and analysed. A number of genes found to be dysregulated in disease compared to controls were confirmed by real-time PCR. This allowed us to identify which method/condition is best suited for carrying out GEP in ALS.RNA was extracted by PAX, TEM and LL as described in the methods Figure 1Averages of RNA yield and RNA quality  for each method were quantified using the Nanodrop and Bioanalyser and are displayed in in vitro transcription (IVT) reaction all samples yielded enough material to progress to the hybridisation step  for the 75 hybridised arrays was carried out. The polyA controls showed that there was no bias between high and low expressed genes during the preparation steps  was used to plot the average expression levels of a number of probe sets in the highest expressing human globin isoforms  for each of the five conditions. In the case of alpha globin probe sets, representative plots (Table S1).Analysis in MAS5.0 was carried out to determine the effect of globin clearance on transcripts present. Analysis of the number of transcripts present in all arrays for each condition Figure 7Using QLUCORE OMICS Explorer, principal component analysis (PCA) on GCRMA normalised samples that took into account the expression levels of all probe sets on the arrays showed that 2 main clusters were visible and broadly corresponding to globin levels . A control analysis was also carried out in order to measure the probability of measuring the number of differentially regulated genes in any two random groups of people of a similar size. This would help identify the amount of noise in our methods. The 15 individuals were randomly sorted into two groups five times (not based on ALS status) and these groups were compared against each other. PUMA analysis carried out for each random group pairing demonstrated the number of differentially regulated genes between groups was lowest in the case of LL with an average of 37.74% ±10.54 of the original number of genes. TEM GC exhibited more noise, with an average of 46.91% ±18.49 decrease compared to the number of genes in the original study (Table S4).A PUMA analysis of differentially-regulated genes between patients and controls was carried out, and the three conditions revealed the following number of statistically significant (p≤0.05) differentially regulated probe sets: LL, 3047; TEM GC, 3619; PAX GC 4511 , with two genes from high, medium and low probability  values were chosen. PCR calculated fold change values were calculated from patient and control ΔCt values Figure 9In the case of LL, 4 out of 6 genes were validated with significance: CDK1, EGR1, SSPN, and IL23R , while 2 genes were not validated as being regulated in the same direction as the arrays: MBLN3 and FOXP1.In the case of TEMPUS/GC, 4 out 6 genes were validated with significance: EP400, E2F2, EHBP1 and KAZ , while one gene was regulated in a similar way to that seen in the array without significance: IGHMBP2 (p = 0.11), and 1 gene, PPP1R10, was not found to be regulated in a similar way observed with the arrays.In summary both methods were able to validate two-thirds (4/6) of the genes chosen thereby indicating that these methods are similarly reliable. However the reliability of a method in terms of gene validation by PCR does not necessarily correlate with the relevance of the method to uncovering genes important in the disease process, therefore gene ontology studies were carried out on the lists of differentially expressed genes identified by the two methods.Initially, functional annotation clustering was carried out using DAVID. In the case of TEMPUS/GC 439 clusters were identified of which the top 15 are ranked Table 3.Although there are similarities between the 2 lists in terms of cluster identification, e.g. nucleus/intracellular organelle, apoptosis/cell death, nucleotide binding, RNA recognition motifs and transcription/transcriptional regulation all being found to be enriched with both methods, there are major differences too. The extent of enrichment in TEM GC for the top 3 clusters: ribosome, nucleus and RNA splicing was much greater than the top 3 clusters for LL  and of the top 3 clusters in TEM GC, ribosome and RNA splicing were not present in the top 15 LL clusters.  Of the top 3 LL clusters, all 3 are found with the top 15 TEMPUS/GC clusters.Tables S5 and S6). KEGG analysis of the LL gene list revealed dysregulation of 45 pathways including a number of signalling cascades such as MAPK, Jak-STAT, and phosphatidylinositol and associated receptor-mediated pathways (Table S5). KEGG analysis of the TEM GC gene list indicated 33 affected pathways (Table S6). Specific signalling cascades observed in the LL list were absent, however a number of common receptor-associated pathways were affected, e.g. Insulin signalling pathway, Neurotrophin signalling, B-cell receptor signalling. Comparing between the two KEGG pathways analyses, LL and TEMPUS share 17 common dysregulated pathways .KEGG pathway analysis was also carried out in DAVID, in order to give an indication of the extent of detected changes in intracellular pathways with each experimental method  and stored at −20°C after the required mixing . In the case of LL, a larger quantity of blood, 8–9 ml, was first collected in a standard vacutainer blood collection tube before being filtered into another collection tube. The LL filter required flushing with PBS and saturating with RNAlater before being stored at −20°C directly. For RNA extraction, using the 2 column-based methods, TEM required less time to carry out compared to PAX, also TEM extraction resulted in high yield, high quality pure RNA without the requirement of DNAse step, which was required for PAX, . LL extraction resulted in intermediate yield but also required less time to carry out than PAX. These observations are in broad agreement with previous studies Globin depletion was carried on PAX and TEM extracted RNA samples using GlobinClear. Levels of the most abundant globin alpha and beta globin isoforms in adult human blood showed reduction in these samples, compared to non-globin cleared samples and averaging lower than levels in LL, but with greater variability. LL samples did exhibit residual levels of globin mRNA, which were also lower than non-globin cleared samples. Previous studies suggested that GC depletion results in lower RNA quality and RIN values in vitro transcription was carried out. IVT yield was greatest in the following order: TEM > LL > PAX > TEM GC > PAX GC. Additionally, while PAX yielded on average more RNA after extraction than LL, the latter method resulted in higher levels of aRNA even though the same amount of input cDNA was used in the IVT reaction. Possible reasons for this may be explained by comparing pre-fragmentation profiles of aRNA observed with the different conditions. Intense discrete peaks at low mass nucleotide ranges could be observed in all TEM and PAX aRNA profiles after Bioanalyzer analysis. All TEM profiles exhibited similar peaks, and all PAX profiles exhibited similar peaks. These probably represent aRNA populations associated with globin since following globin depletion these peaks disappeared in all cases and a wide range of RNA populations were observed in both TEM and PAX. A similar broad range of aRNA populations were visible in pre-fragmented LL samples. After fragmentation, profiles were indistinguishable between methods. These observations highlight the importance of globin depletion/reduction in RNA extracted from blood samples, as aRNA populations from globin skew the relative proportions of other aRNA populations within samples.All methods/conditions yielded sufficient quality and quantity of RNA to proceed with a reverse transcription reaction. After second strand synthesis, amplification and production of aRNA by Quality control metrics were consistent within each sampling method. However, when comparing between methods the saturating presence of globin significantly affected the percentage of present calls using MAS5 analysis. PAX and TEM present calls were distinctly lower than LL, PAX GC and TEM GC. Thus, already we can distinguish two separate groups of arrays, a “globin positive” group with low % present calls/large scale factor, and a “globin negative” group with higher % present calls/low scale factor. The identity of these 2 groups is highlighted in the PCA plot in Using the PUMA bioconductor package Due to the technical limitations of the PAX technique , this method was disregarded for PCR validation which was carried out on LL and TEM GC samples, as they represent better methods for GEP profiling. Six genes were chosen with a minimum of 1.5 fold difference between disease and controls for each method and real-time PCR resulted in the validation of 4 out of 6 genes for each condition, indicating that both methods present similar consistency and reliability. These similarities in consistency between the 2 methods may not represent similarities in differential regulation of gene pathways, and therefore GO functional annotation clustering analysis was carried out on the respective gene lists to ascertain putative differences/similarities. Considering the a) relatively few differentially regulated genes in common between the 2 methods, and b) the difference in starting material (whole blood versus leukocyte enriched), GO analysis revealed a striking similarity in the number of common cluster terms, and this was supported by similar observations when carrying out KEGG pathway analysis. It is noteworthy that the top cluster terms, ribosome-associated genes, in TEM GC were not present in LL, and this raises the question as to whether these are associated with pathological changes and/or mirror underlying differences in initial cell populations used as starting material for each extraction method. As there are only small differences between globin levels (depleted and undepleted isoforms) in TEM GC samples when comparing patients and controls (data not shown) we can discount any potential sampling/extraction bias leading to the observed differences in ribosome-associated gene expression. Taking into account the relatively small number of ALS patients and controls in this study, drawing conclusions regarding pathway analysis should be cautious, however examples of pathways involved in ALS include: defects in RNA processing GEP with blood is a feasible approach for biomarker discovery in neurological disease. Overlap in transcript expression and splicing between blood and neural tissues has been demonstrated 2EDTA spray-coated collection (BD Vacutainer) tubes (approx. 8–9 ml), for immediate passage through a LL filter. RNA was stabilized in PAX and TEM tubes by slow inversion (PAX) or vigorous shaking (TEM) for 30 and 15 seconds respectively. LL filters were washed with PBS and saturated with RNAlater as described in the manufacturer's instructions. Samples were stored at −20°C until processed further.Ethical approval for the study was granted from South Sheffield Ethics Committee and written informed consent from each subject was obtained before commencing. Blood was collected after an overnight fast from patients (n = 8) or age- matched controls , TEM (Applied Biosystems) and LL (Ambion) methods as described in the respective maufacturer's instructions, in each case omitting any DNase step. All samples were extracted separately and not pooled. The quality and yield of extracted RNA was analysed by examining electropherogram traces generated by the 2100 Bioanalyzer (Agilent) running the samples on a total eukaryote RNA nano chip, and by UV spectrography using Nanodrop 1000 (Thermo Scientific). Samples were stored at −80°C until required for globin depletion (TEM) or amplification (LL). Before globin depletion was carried out on TEM and PAX samples, PAX samples underwent DNase (NEB) treatment  for 10 mins at 37°C followed by addition of EDTA to a final concentration of 5 mM and subsequent heat inactivation at 75°C for 10 mins. Samples were run on the 2100 Bioanalyzer to measure quality, and subsequent globin depletion  was carried out with 1 µg of RNA as described in the manufacturer's instructions. Where the RNA concentration was <67 ng/µl, samples had to be concentrated using 50 µg/ml Glycoblue (Ambion), in 0.5 M ammonium acetate, 50% isopropanol.in vitro transcription reaction. In order to minimise batch effects, 10 samples were randomly chosen for each round of IVT. A number of samples had to be concentrated using 50 µg/ml Glycoblue (Ambion), in 0.5 M ammonium acetate, 50% isopropanol before fragmentation. 15 µg of aRNA was fragmented as described in the manufacturer's instructions and hybridized overnight onto U133 Plus 2.0 human genome arrays as previously described Linear amplification was employed using the Affymetrix GeneChip 3′ IVT Express Kit according to the manufacturer's instructions. 200 ng of total RNA was reversed transcribed to synthesize first-strand cDNA with oligo(dT) primers containing a T7 flanking sequence. After second strand synthesis, labelled aRNA was synthesized in an Quality control was initially carried out on Affymetrix expression console, on MAS5.0 normalised data. The data were also normalised by GCRMA for further quality control, gender differences using the program QLUCORE OMICS Explorer and analysis of globin isoform levels in GENESPRING (Agilent). CEL file data were analysed by the R package PUMA for compiling a list of differentially regulated genes. We utilised PPLR (probability of positive log-ratio), a tool within the PUMA suite that measures the uncertainty in estimation of gene expression, i.e. the technical variability, in order to provide a robust estimate of differential expression and listed significantly differentially regulated genes with a pLikeValue <0.05 . Gene Ontology and KEGG pathway analysis was carried out using the bioinformatics tool DAVID −(ΔΔCt). Unpaired two-tailed t-tests were used to determine whether differences in gene expression levels between patients and controls were statistically significant.200 ng of total RNA from 6 of the patients and 6 of the controls Table 5 Table S1Gene Ontology of genes unmasked by globin depletion (top 100 GO terms).(XLSX)Click here for additional data file.Table S2List of statistically significant  differentially regulated genes after PUMA analysis of LL samples .(XLSX)Click here for additional data file.Table S3List of statistically significant  differentially regulated genes after PUMA analysis of TEMPUS GC samples .(XLSX)Click here for additional data file.Table S4Control analysis: the number of differentially regulated genes for each of the 5 random pairs of groups after PUMA analysis and the number of genes expressed as a percentage of the original study. The percentages were averaged and standard deviation was also calculated (STDEV). The distribution of ALS patients between groups is also detailed.(XLSX)Click here for additional data file.Table S5KEGG pathway analysis of LL affected pathways.(XLSX)Click here for additional data file.Table S6KEGG pathway analysis of TEM GC affected pathways.(XLSX)Click here for additional data file.


Clinically, we found that increased expression of mdig in cancer tissues correlates with poorer overall survival of the lung cancer patients, esp., for those without lymph node metastasis. Taken together, our results suggest that mdig plays opposite roles on cell growth and motility, which possibly indicates the paradoxical effect of mdig at the different stages of carcinogenesis.Increased expression of mineral dust-induced gene  has been observed in a number of human cancers. The mechanism of how mdig contribute to the pathogenesis of cancer remains to be fully elucidated. In this report, we demonstrated that overexpression of mdig decreased the nuclear staining signal by 4′,6-diamidino-2-phenylindole (DAPI), along with a considerable enhancement in cell proliferation. Silencing mdig by shRNA resulted in a statistically significant decrease of cell proliferation. Intriguingly, mdig overexpression reduced the capacity of the cells in migration and invasion  Although these features are mutually correlated, each of these features is governed by different biochemical and intracellular signaling pathways. In the earlier stages of carcinogenesis, sustained proliferation is the key for the formation of detectable tumor mass that interferes with the normal functions of the given organs or tissues. During tumor progression, heterogeneous tumor cell subclones arises through genetic and epigenetic evolution of new tumor cell lineages that are able to replenish the cancer cell population and propagate the cells to distant sites The basic features of cancer cells include fast proliferation, migration and invasion We had previously identified a mineral dust-induced gene (mdig) in alveolar macrophages isolated from the people with chronic lung diseases resulted from the occupational exposure to mineral dust in mining industry in vitro. Overexpression of mdig in lung cancer cell line, A549, provides the cells with growth advantage. However, at the same time, these cells exhibited reduced potential of invasion and migration.In the present report, we demonstrated that mdig has different roles in cell proliferation and invasion/migration nd row, Earlier studies suggested that mdig/mina53/NO52 is predominantly localized in the nucleolar compartment of the cells The intracellular localization of the endogenous mdig was also determined for the cells at different cell cycle phases. In interphase cells , the endogenous mdig was mainly found in the nucleoli of the cells (3+), a typical stress inducer, for 0.5 to 6 hours (h) followed by RT-PCR to measure the levels of mdig mRNA and immunofluorescent staining for the determination of intracellular distribution of the mdig protein in these cells. As depicted in 3+ was able to induce the accumulation of mdig mRNA that was peaked at 4 h and remained high at 6 h. Immunostaining of mdig revealed that at 1 h of As3+ treatment, mdig was mostly localized in nuclei. After 2 to 6 h treatment, some cells showed a punctuation or speckled pattern of the mdig localization in the cytoplasm. These punctuated mdig staining signals were not uniform in size. Although As3+ treatment appears to be able to increase the number of stress granules, most of the punctuated mdig staining signals were not overlapping with these granules  stably expressing a deletion-mutated mdig in our previous studies To explore whether the above findings are clinically relevant for the lung cancer patients, we analyzed mdig expression level and the survival data of 1,715 lung cancer patients using an online gene profiling database by stratifying patients based on the higher or lower mdig expression A number of human cancers exhibited increased expression of mdig gene, including lung cancer The opposite effects of mdig on cancer cell proliferation and motility or invasion may be clinically relevant for the pathogenesis of the lung cancer and the prognosis of the lung cancer patients. Analyses of the mdig expression levels and the patient survival data clearly suggest that increased expression of mdig predicts poorer overall survival (OS) of the total lung cancer patients  and the By studying mdig/mina53 expression in lung cancer tissues from 101 patients and correlating the mdig expression with the patient survival time, Komiya et al. Since its first discovery from human lung alveolar macrophages 2. For cell transfection, 5×105 cells were seeded in 6-well plates. Cell transfection was performed when the cells reached to 60–70% confluence using Lipofectamine 2000 (Invitrogen). The mdig-GFP expression vector, control vector, RFP-conjugated mdig shRNAs, and RFP-conjugated control shRNA were purchased from OriGene . For establishing stably transfected clones, the cells were cultured in the media containing 2 µg/ml of puromycin for 3 weeks. The GFP- or RFP-positive clones were maintained for subsequent analyses of cell proliferation, migration, invasion, and additional immunofluorescent staining or Western blotting.Human lung adenocarcinoma epithelial cell line A549 and human bronchial epithelial cell line BEAS-2B are purchased from the American Type Culture Collection . The cells were maintained in RPMI1640 or DMEM medium  supplemented with 5% fetal bovine serum and 1% penicillin-streptomycin  in 37°C humidified incubator in the presence of 5% COTotal cellular proteins were prepared by lysing the cells in RIPA buffer  supplemented with phosphatase/protease inhibitor cocktail and 1 mM PMSF through sonication and centrifugation, followed by quantification using a Micro BCA Protein Assay Reagent Kit . Before loading on 10% SDS-PAGE gels, the proteins were boiled in LDS sample buffer (Invitrogen) containing 1 mM dithiothreitol. The separated proteins were transferred onto PVDF membranes (Invitrogen) and subjected to immunoblotting with the indicated antibodies.5′-CTG AAC GGG AAG CTC ACT GGC ATG GCC TTC-3′ and 5′-CAT GAG GTC CAC CAC CCT GTT GCT GTA GCC-3′.Total RNAs were extracted from the cells using TRIzol Reagent and subjected to RT-PCR using AccessQuick RT-PCR system purchased from Promega . The PCR primers for mdig are: 5′-TCA TGT CGG GCC TAA GAG AC-3′ and 5′-GGC ATT TGA TTC TGC AAA GG-3′, which amplifies a 1,510 bp DNA fragment covering the whole coding region of the mdig gene. PCR primers for GAPDH are: 3/per well in 500 µl medium in 96-well plates. At the indicated time points after seeding, 20 µl of MTT solution  was added to each well and incubated at 37°C for another 3.5 h. At the end of incubation, the medium was replaced with 150 µl of DMSO (Fisher) to dissolve the formazan crystals. After agitation on shaker for 15 min, absorbance at 570 nm–690 nm was measured using a Biokinetics plate reader Cell proliferation rate was determined by seeding 5×10Cell migration and invasion were determined using BD BioCoat™ Matrigel™ Invasion and Migration Chambers according to the manufacturer's instruction. After incubating for 24 h (migration) or 48 h (invasion), the cells in the upper chambers were scrubbed out using cotton tipped swab. The cells on the lower surface of the membrane were stained with the Diff-Quik Kit. The migrated and invasive cells were counted under a microscope.p values of <0.05 between mdig higher (mdighigh) and mdig lower (mdiglow) groups were considered significantly different. The best performing threshold (best cutoff) was used as a cutoff of the survival data.A Kaplan-Meier survival database that contains survival information of 1,715 lung cancer patients and gene expression data obtained by using three different versions of Affymetrix HG-U133 microarrays was used


Bbs4;Mks1 double mutant embryos show a stronger phenotype than either single mutant, with completely penetrant micropthalmia and polydactyly, loss of ventral neural cell types, and a reduced number of short bulbous primary cilia. This additive phenotype suggests that BBS and MKS function in parallel pathways. We detected accumulation of Smo and abnormal Gli2 localization in Bbs4;Mks1 double mutant cilia, suggesting that the double mutants have a defect in retrograde trafficking. To define a role of BBS4 or MKS1 in ciliary trafficking, we have generated Bbs4 or Mks1 mice that carry mutations in the IFT machinery. Absence of BBS4 did not affect the phenotype of a weak IFT-B mutant or of embryos that lack the retrograde dynein motor, whereas the Mks1 phenotype was stronger when either IFT-B or dynein were mutant. This implies that BBS works together with IFT to regulate ciliary trafficking whereas MKS1 has a parallel role in ciliary function. Together these data suggest that BBS and MKS both have roles in regulating movement of Hh components in the cilium however the way in which they do this is distinct from each other.Bardet-Biedl syndrome (BBS) and Meckel-Gruber syndrome (MKS) are genetic disorders caused by disruption of primary cilia. The BBS and MKS proteins are found in two distinct protein complexes but the functional relationships between these two complexes are not clear. Here we analyze double mutant mouse embryos to define these relationships.


MTAP) is a tumor suppressor gene that is frequently inactivated in a wide variety of human cancers. In this study, we have examined if heterozygosity for a null mutation in lacZ)Mtap . Functional categorization of these genes reveals enrichments in several pathways involved in growth control and cancer.Survival in both models was significantly decreased in Mtap allele alters gene expression and enhances lymphomagenesis in Eµ-myc mice.Our findings show that germline inactivation of a single  MTAP is a metabolic enzyme in the methionine salvage pathway that converts the polyamine synthesis byproduct 5′-dideoxy-5′-methylthioadenosine (MTA) into adenine and methylthioribose-1-phosphate and is expressed in all tissues throughout the body MTAP gene is frequently inactivated in human tumors by large homozygous deletion of the 9p21 region where both the MTAP and the CDKN2A/ARF tumor suppressor genes are located CDKN2A/ARF as well as MTAP, it was initially hypothesized that loss of MTAP in tumors was simply due to it being a co-incident bystander. However, a variety of studies now indicate that MTAP is a tumor suppressor gene in its own right. Re-expression of MTAP protein in MTAP-deleted MCF-7 breast adenocarcinoma cells results in loss of anchorage independent growth in vitro and the ability to form tumors when injected into SCID mice MTAP-deleted melanoma cell line or gastric cancer cell line results in reduced invasion and migration in vitroMTAP gene (Mtap) die prematurely of T-cell lymphoma with a mean age of onset of about 18 months MTAP functions as a tumor suppressor gene independent of CDKN2A/ARF.The MTAP enhances tumor formation involves the link between MTAP and polyamine metabolism. Polyamines are small aliphatic amines essential for cell growth and are elevated in tumors MTAP results in up-regulation of ODC in both yeast and human cells in vitro and cause increased frequency of skin tumors in a transgenic mouse model Eµ-myc transgenic mouse model of lymphoma and that this over-expression was important for lymphomagenesis Myc oncogene.A potential mechanism by which loss of lacZ/+Mtap mice with both Eµ-myc mice and +/−Pten mice and have characterized the offspring for tumor formation. There were three distinct goals of these studies: 1) Strengthen the hypothesis that Mtap-loss plays a functional role in tumor formation; 2) Create a mouse model to study the effects of Mtap-loss on tumor formation that had a significantly shorter latency period than the original lacZ/+Mtap strain; and 3) Test the hypothesis that loss of Mtap might accelerate tumorigenesis in Eµ-myc mice by causing over expression of ODC.In the studies described here, we have crossed Mtap mice were created and genotyped as previously described Eµ-myc mice were obtained from the lab of Dr. John Cleveland (Scripps) and genotyped as described in Pten mice were provided by Dr. Antonio Di Cristofano (Albert Einstein University) and genotyped as described in Eµ-myc animals, animals were monitored for survival and tumor formation daily by visual inspection and palpation. In these animals, tumor formation was obvious as indicated by swelling around the neck and associated lethargy. When tumor or distress was detected, the animals were euthanized and necropsied. +/−Pten animals were monitored in a similar manner, but sometimes the animals died spontaneously without tumors being detected. In cases where the deceased animal was relatively fresh, necropsies were performed to determine if tumor was present at time of death. In cases where a tumor could not be confirmed at the time of necropsy, animals were censored for the purposes of survival analysis.For All animal protocols were approved by the Fox Chase Cancer Center IACUC (Protocol #05-06) and done in compliance with NIH guidelines. Animals were monitored daily for signs of distress and suffering. If distress or tumors were detected, animals were euthanized by overdose with isoflurane.µl. For each, visualization was achieved by incubation followed by incubating with either a goat anti-rabbit or anti-rat biotinylated secondary antibody followed by incubation with streptavidin peroxidase and 3,3′-Daminobenzidine (Sigma-Aldrich) substrate chromogen. Each slide was assessed blindly by a by a trained pathologist specializing in lymphoma (T.A-S) using a 1–6 grading system in which the percentage of cells containing the graded feature  was determined. A grade of 1 equals <10% of cells testing positive, 2 = 10–30%, 3 = 30–50%, 4 = 50–70%, 5 = 70–90%, and 6<90%.Autopsied materials were fixed in buffered formalin, embedded in paraffin, and processed as previously described FACS analysis on spleen from euthanized animals was performed as previously described TdT and Cµ analysis, total RNA was prepared by sorting 105 cells into “Solution D,” followed by cDNA preparation as previously described ß-actin as an internal control.For −.Mtap protein levels were detected by Western blot analysis using a MTAP monoclonal antibody (Santa Cruz Biotechnology) at a 1/1000 dilution. Signal was visualized by SuperSignal West Pico Chemiluminescent kit (Pierce), and signal was quantified using Alpha Innotech image analyzer. All levels were normalized to an alpha-actin internal control. Mtap expression <20% that of control samples was scored as Mtap+/+Mtap and lacZ/+Mtap mice were excised and put into RNAlater (Ambion). Total RNA was isolated using TRIzol reagent  and further purified by RNeasy kit  according to manufacturer’s instructions. RNA quality was evaluated by electrophoresis on Agilent Bioanalyzer (Agilent). Five µg of total RNA was first transcribed into cDNA using the Invitrogen’s Superscript system with an oligo (dT)24 primer containing a T7 RNA polymerase promoter sequence at its 5′ end. After double stranded cDNA synthesis using DNA polymerase I, Biotin-labeled cRNA was generated by in vitro transcription using a GeneChip IVT Labeling Kit (Affymetrix) according to manufacturer’s instructions and then purified using the GeneChip Cleanup Module. Label target was fragmented to a size of 35–200 bases by metal-induced hydrolysis prior to hybridization. Target hybridization was performed in an Affymetrix hybridization oven at 45°C for 16 hours using an Affymetrix GeneChip Mouse Genome 430A 2.0 Array. After hybridization arrays were washed using the Affymetrix fluidics station and stained with streptavidin Phycoerythrin according to the Affymetrix protocol. Four bacterial and phage cRNA controls  were included in the hybridization buffer to serve as internal control for hybridization efficiency. Washed arrays were scanned on an Affymetrix GeneChip Scanner 3000. Data was normalized using RMA as previously described Livers from 100-day-old male http://www.ingenuity.com). Both the Web Gestalt and IPA software maps the enriched genes on various canonical pathways and determines if the number of hits in each pathway exceeds those estimated by chance. The Web Gestalt software gives both an unadjusted and an adjusted P-value, where the adjusted P-value represents the false discovery rate. The IPA software ranks each pathway  by a score that is the negative log of the P-value. The IPA software does not determine a false discovery rate.For pathway analysis, we selected a set of differentially regulated genes based on the criteria that they exhibited at least a 50% change in mRNA levels and had a p-value <0.01 (FDR <0.29). This list, containing 363 probes, was then analyzed using both Web Gestalt Gene Analysis Toolkit V2 http://www.graphpad.com). For comparison of continuous variables, a Student’s t-test (2-sided) was used.P-values for contingency tables (2×2) were all performed using Fischer’s exact test (2-sided). Survival curve comparisons were assessed using the log-rank test supplied in GraphPad Prism 4.0  is a phosphatase that dephosphorylates phosphatidylinositol -trisphosphate resulting in the formation of phosphatidylinositol -biphosphate, which causes inhibition of the AKT signaling pathway. Germline mutation in PTEN in humans is associated with Cowden’s disease, characterized by the presence of cutaneous benign hamartomas and high frequency of thyroid and breast cancer Pten in mice is embryonic lethal, but heterozygous Pten mice (+/−Pten) develop a variety of tumors including lymphomas, endometrial tumors, colon cancer, and gonadal tumors that are detectable between six months and one year of age +/−Pten mice with lacZ/+Mtap animals and followed a cohort of +/+ Pten+/−Mtap and lacZ/+ Pten+/−Mtap for up to 450 days. We observed significantly decreased tumor-free survival in lacZ/+Mtap animals compared to +/+Mtap . These results show that heterozygosity for Mtap decreases survival in +/−Pten animals.We also examined a second mouse tumor model,  animals . The mos animals . Other tEµ-myc mice. First, we examined thymus sections from control, +/+,Eµ-myc Mtap and lacZ/+Eµ-myc Mtap animals for a variety of morphological and immunohistochemical features. As expected, staining with anti-bodies to either CD3 (T-cell marker) or CD45R/B220 (B-cell marker) indicated that all the lymphomas in both +/+Eµ-myc Mtap and  +/−Eµ-myc Mtap animals were B-cell neoplasms (not shown). Morphologically, these lymphomas exhibited a spectrum between large cells, with irregular nuclear membranes, vesicular chromatin, and prominent nucleoli (diffuse large B-cell lymphoma-like) to others with medium sized cells, relatively fine chromatin and small nucleoli with brisk mitotic activity and apoptosis resembling Burkitt’s lymphoma. Some fell in between resembling “grey zone” lymphoma  expression by the pro B cells − plasmacytoma. Taken together, our data show that the cell of origin of the lymphomas was most likely started from the pre-B stage of development in both +/+Mtap and −/−Mtap animals.Because of both the earlier appearance and the increased grade of the tumor, our next question was whether lacZ/+Mtap and 17 +/+Mtap animals by Western blot analysis (lacZ/+Mtap mice showed complete loss of MTAP protein compared to 5/17 (29%) of the tumors from +/+Mtap mice, but this difference was not statistically significant . Of these, 242 were up regulated and 121 were down regulated in enes see . We searenes see . Anotherenes see .We also subjected the same list of to analysis by the IPA software. The top five networks identified were: 1) Lipid Metabolism, Molecular Transport, Small Molecule Biochemistry (score 44); 2) Cancer, Endocrine System Disorders, Hematological Disease (score 31); 3) Cell Morphology, Cancer, Developmental Disorder (score 29) 4) Humoral Immune Response, Protein Synthesis, Hematological System Development and Function (score 25); and 5) Cell-To-Cell Signaling and Interaction, Skeletal and Muscular System Development and Function (score 25). A list of the cancer related genes identified by IPA is shown in Mtap allele may have protumorigenic affects.The finding of a significant number of cancer related genes in the differentially regulated gene set is consistent with the idea that loss of a single Sat1) were increased in lacZ/+Mtap animals, while the biosynthetic gene spermidine synthase (Srm1) was down regulated.We also examined transcripts of genes known to be involved in polyamine biosynthetic and degradation pathways . We founMtap died of T-cell lymphoma with a median life expectancy of 18 months Mtap is a bona fide tumor suppressor gene, the long latency of this model makes it impractical for more extensive studies. An additional disadvantage of this model is that often lacZ/+Mtap mice would die of disease without exhibiting obvious external symptoms, making it difficult to get preserved tissue to study. To circumvent these problems, and to further establish that Mtap has tumor suppressor activity, we examined if a germline mutation in Mtap could cooperate and accelerate tumorigenesis in two other mouse tumor models, Eµ-Myc and +/−Pten. These models were chosen both because they have well defined tumor types and because they both have been used successfully to identify genetic interactions with other tumor suppressor genes such as p53, ARF, and CDN2K Despite the knowledge that loss of MTAP expression is a relatively frequent event in a variety of different tumor types, the biological importance of MTAP loss in tumorigenesis has only recently started to be addressed. An important motivation for the studies described here was our earlier study in which we showed that mice heterozygous for Mtap decreases tumor free survival in Eµ-Myc mice, with the median time for detectable tumor formation or death decreasing by 33%. For Pten+/− mice, we did observe reduced survival, but did not observe a statistically significant increase in tumor formation the necropsied lacZ/+ Pten+/−Mtap animals. The reason for this apparent contradiction is that a larger percentage of the MtaplacZ/+ animals died spontaneously and the samples were too badly decayed to be necropsied. Whether these animals died of tumors cannot be definitively determined. In retrospect, a proactive necropsy done at a particular time point probably would have been a superior strategy. On the other hand, lacZ/+Eµ-myc Mtap mice developed rapidly forming tumors that were easily detected by observing swollen lymph nodes in the neck of the effected mice. Histopathologic and FACS analysis of the lymphomas indicate that the cells of origin are pre-B and immature B cells, and that this cell type was the same for both +/+Mtap and lacZ/+Mtap animals. This finding indicates that lacZ/+Mtap can cooperate with myc in driving lymphoma formation and that lacZ/+Mtap does not alter the developmental stage of the cells giving rise to the lymphoma. However, we found that the tumors from lacZ/+Mtap animals were of a higher grade as judged both by cell morphology and staining for the proliferation marker Ki67. This, along with the earlier appearance of the tumors, suggests that loss of Mtap may cause increased tumor aggressiveness.Our data clearly show that heterozygosity for Mtap expression was lost in the lymphomas developed in Eµ-myc Mtap mice. We found that 5/17 tumors (29%) from +/+Mtap mice had lost Mtap expression compared to 13/26 (50%) from the lacZ/+Mtap animals. Although the frequency of Mtap- tumors appeared to increase in lacZ/+Mtap animals, this increase was not statistically significant and is unlikely explain the dramatic decrease in latency time observed in the lacZ/+Mtap animals. Rather, this data suggests that Mtap may be acting in a haploinsufficient manner. To develop evidence that germline heterozygosity for Mtap can have phenotypic consequences, we performed microarray experiments examining gene expression profiles in the livers of young age and sex matched +/+Mtap and lacZ/+Mtap animals. Based on the skewed distribution of P-values of the probes, we estimate that as many as 2048/16716 probes examined (14.4%) may be differentially expressed. Confining ourselves to probes that show at least a 50% difference in expression levels, we identified at least 363 probes representing 251 unique genes. These genes include many genes involved in pathways implicated in cancer development and progression. Because these experiments were done using RNA derived from liver, it is unclear if the genes and pathways identified as being affected by Mtap are directly relevant for the accelerated lymphoma development in these animals. Nonetheless, these experiments clearly show that loss of a single Mtap allele can have significant biological effects.We also examined the frequency by which Mtap and an up-regulation of ODC, a key enzyme affecting polyamine metabolism lacZ/+Eµ-myc Mtap mice tended to have higher levels of ODC expression than tumors found in +/+Mtap animals. In addition, we found Mtap-dependent differences in the liver mRNA levels of two polyamine metabolic genes (Sat1 and Srm1). Taken together, these observations provide additional support that Mtap-loss affects polyamine metabolism. A possible mechanism by which elevated ODC may contribute to lymphomagenesis may be via its influence on apoptosis. In hematopoietic cell lines, high levels of ODC have been shown to suppress apoptosis by reducing intracellular ROS species Mtap might also promote lymphomagenesis by other means as well. In unpublished studies, our lab has found that expression of Mtap in an Mtap deleted osteosarcoma cell line can suppress several tumor related phenotypes without any effect on ODC levels . Thus, it seems possible that there may be multiple mechanisms by which Mtap-loss promotes tumor formation.Previous studies have shown a relationship between loss of Mtap can cooperate genetically with at least two other cancer causing mutations, Eµ-myc and +/−Pten, to reduce survival and, in the case of Eµ-myc, accelerate tumorigenesis. This acceleration does not appear to require the loss of the wild-type Mtap allele, suggesting that loss of a single copy of Mtap may have protumorigenic affects. Consistent with this view is the observation that heterozygosity for Mtap results in large alterations in the liver gene expression profile. Our findings support the view that Mtap-loss is of biological importance in tumorigenesis.In summary, we have shown here, for the first time, that germline mutations Table S1Mtap differentially expressed genes.(XLSX)Click here for additional data file.Table S2Gene Ontology Pathways affected by Mtap.(XLSX)Click here for additional data file.Table S3Kegg Pathways affected by Mtap.(XLSX)Click here for additional data file.Table S4Cancer genes identified by IPA analysis.(XLSX)Click here for additional data file.Table S5Analysis of Polyamine Pathway genes.(XLSB)Click here for additional data file.


Patients with end-stage renal disease can easily acquire a hepatitis C virus (HCV) infection via several ways. An HCV infection is difficult to treat after renal transplantation due to the conflicting actions of immunosuppressant therapy to maintain the function of the transplanted kidney and viricidal interferon (IFN) or ribavirin (RBV) treatment. Antiviral therapy requires great caution to avoid the complex and potentially fatal pharmacological effects. In this review, we examined clinical challenges and potential solutions for this specific scenario.We searched Pubmed (NLM), LISTA (EBSCO), Web of Science (TS). The management of patients on waiting list, the indications and regimens about treatment were studied.More than forty papers about this topic were found, including seven small clinical trials. International consensus has been reached to test patients awaiting renal transplantation. HCV detection after renal transplantation warrants careful consideration of when to initiate antiviral therapy. Treatment will begin immediately if deteriorating liver function increases the risk for loss of renal function. The choice of regimen depends on the patient's renal function and is individualized under close observation. The immunosuppressive regimen will be adjusted accordingly before antiviral therapy is initiated.The effects of modified antiviral therapy on these patients varies because of individual characteristics and disease state, and also because of the difficulty associated with conducting a large clinical trial to obtain statistically sound conclusions. The management before transplantation is important and when antiviral therapy needs to start, careful consideration of risks and benefits is needed before initiating this type of treatment. Patients with end-stage renal disease (ESRD) are required to undergo planned hemodialysis before renal transplantation is considered . DialysiTransplanted kidneys usually maintain their function with continuous administration of immunosuppressants. Commonly used immunosuppressive regimens include the tacrolimus FK506) regimen (FK506 + mycophenolate mofetil [MMF] + prednisone) and the cyclosporine A (CsA) regimen (CsA + MMF + prednisone) . These r06 regimePubmed (NLM), LISTA (EBSCO), Web of Science (TS) were searched with key words "Renal Transplantation", "Hepatitis C Virus", "Antiviral Agents", and "Immunosuppression" in recent 10 years, mostly recent 5 years. We also tried to obtain full articles and abstracts on the reference lists from retrieved documents. As the information about this topic was rare, small clinical trials and case reports were also included. The complexity of antiviral therapy, the management of patients on waiting list, the indications and regimens about treatment were studied.We found more than 40 papers including 7 small clinical trials about this topic. From the information we drew out following aspects.The treatment of HCV infection demands immune enhancement while kidney anti-rejection therapy requires immune suppression. An international consensus has been reached to conduct HCV testing on patients awaiting renal transplantation . A similOne third of ESRD patients with HCV infection can achieve SVR via IFN administration, and thus, antiviral therapy is recommended for those patients. The combination of IFN with ribavirin (RBV) has further improved the rate of SVR. After treatment, patients who have attained SVR usually exhibit a long-term and durable virology, biochemistry, and histology response . SeveralAlthough effective screening and treatment strategies have been implemented to help avoid dysfunction of the transplanted kidney, a big challenge in recrudescence and reinfection with HCV exists due to the side effects of IFN and RBV, which lead to the inability for most ESDR patients to accomplish the entire standard therapy regimen identified as "888." Patients also risk HCV infection from protracted blood exposure to externalities during renal transplantation surgery.The questions of whether an antiviral treatment can be initiated and how to implement remain controversial. In 2009, the guidelines regarding the diagnosis and treatment of hepatitis C, which were jointly amended by the American Association for the Study of Liver Diseases (AASLD), Infectious Diseases Society of America (IDSA), and American Society of Gastroenterology (ASG), declared that antiviral therapy based on IFN is not recommended for use in patients after renal transplantation due to the risk of acute rejection. This statement applies to patients with chronic kidney disease or to patients following organ transplantation. However, for patients with fibrosing cholestatic hepatitis, IFN is necessary for slowing liver cirrhosis and extending long-term survival 22]23].[23].22][.[23].22]To summarize these studies and guidelines, several major points regarding when to initiate antiviral therapy in renal transplant recipients infected with HCV are listed below:1) Renal transplantation recipients with stable renal and liver function require strict observation rather than antiviral therapy. Liver and renal function tests should be conducted every 3 months and the viral load should be evaluated every 6 months. In particular, a liver biopsy should be repeated every 3 years .2) Patients exhibiting fibrosing cholestatic hepatitis, advanced liver fibrosis, or HCV-associated glomerulonephritis should initiate antiviral therapy because it has known effects on long-term survival rates of both patients and allografts. Sustained suppression of necroinflammation may result in regression of cirrhosis which, in return, leads to decreased morbidity.3) If antiviral therapy is deemed necessary, clinicians should assess first the risks and benefits and then inform the patients. Treatment initiation can only occur with both patient agreement and determination that the benefits outweigh the risks.4) Because liver and renal function, the state of immunosuppression, and the maintenance regimen differ from patient to patient, as do the HCV complications and the impact of antiviral drug side effects, treatments should be individualized. All of these components require close evaluation before final decisions regarding antiviral treatment can be made.In addition, the risk for acute rejection is higher during the first year after transplantation surgery . Thus, i3.3.1. Standard RegimenThe 2009 edition of the diagnostic and treatment guidelines (AASLD/IDSA/ASG) for hepatitis C suggest that the standard treatment should be a combination of pegylated IFN α (PEG-IFN α) and RBV. The specific dosage and duration differ according to viral genotypes and patient response. Patients infected with HCV genotype 1 or 4 should expect a 48-week treatment duration consisting of 180 μg PEG-IFN α-2a per week, administered by hypodermic injection, and a daily weight-dependent dose of RBV, i.e. 1000 mg (body weight ≤ 75 kg) or 1200 mg (body weight > 75 kg). If PEG-IFN α-2b is used, a subcutaneous dose of 1.5 μg/kg should be administered weekly in addition to daily weight-dependent RBV, i.e. 800 mg (weight < 65 kg), 1000 mg (weight 65~85 kg), 1200 mg (weight 85~105 kg), or 1400 mg (weight > 105 kg). Patients infected with HCV genotype 2 or 3 should expect a basic course of 24 weeks with 800 mg RBV daily. If a patient cannot tolerate the side effects, 3 MU of IFN can be used 3 times per week. In addition, the dose of PEG-IFN α-2a can be reduced to 135 μg per week and PEG-IFN α-2b to 1μg/kg per week. Combined RBV is still acceptable when reduction and side effects such as anemia are closely monitored .3.3.2. Reformed RegimenWhat we had identified as stable renal function after transplantation was in fact not actually intact; non-impaired renal function includes either intact renal function or mild to moderate impairment due to HCV-related hemolysis, EMC, or other complications that are not in advanced stages 28]. Thu. Thu28].Combined treatment is applicable for patients with complete renal function. According to actual patient glomerular filtration rates (GFR), RBV is used to supplement the recommended IFN dose. The ultimate goal is to achieve a blood concentration of 10-15 μmol/L over the long term. A trial was conducted previously that used a low-dose combination therapy consisting of the common 1 MU of IFN 3 times per week plus 600 mg of RBV per day . The regBefore initiating antiviral therapy, the immunosuppression regimen should be adjusted accordingly. When a patient is confirmed with HCV infection, it is reasonable to reduce the dosage of immunosuppressants to the lowest dose at which an immunosuppressive state can be maintained and liver damage is minimized. MMF metabolites can inhibit HCV replication effectively; thus, an improved long-term survival rate is achieved in patients without administering antiviral treatment. Further studies are needed to demonstrate how MMF metabolites are processed and removed when used alongside antiviral therapy. All current immunosuppressive regimens include MMF . In animBecause of the specificity of patients and disease state, no consensus has been reached on the effect of modified antiviral therapy on renal transplantation patients, and large clinical trials to obtain statistically sound conclusions are difficult to conduct due to the unique nature of this disease. Nonetheless, several small trials have reported satisfactory curative effects, i.e. the SVR reached 62% with no harm to renal function . OverallTwo phase II, multicenter studies of TVR combined with PEG-IFN α-2a and RBV in untreated patients infected with HCV genotype 1 resulted in the addition of TVR and significantly increased rates of SVR, albeit with enhanced discontinuation due to adverse events 43]. Ras. Ras43].However, the actual effects of upcoming protease inhibitors will be examined in a special group of patients. Highly anticipated RNA interference therapies still remain in the research and development stage . Given t