Hepatic cirrhosis is a common pathway of progressive liver destruction from multiple causes. Iron uptake can occur within the hepatic parenchyma or within the various nodules that form in a cirrhotic liver, termed siderotic nodules. Siderotic nodule formation has been shown to correlate with inflammatory activity, and while the relationship between siderotic nodule formation and malignancy remains unclear, iron distribution within hepatic nodules has known implications for the detection of hepatocellular carcinoma. We aimed to evaluate the role of abdominal susceptibility-weighted imaging in the detection of siderotic nodules in cirrhotic patients.Forty-six (46) cirrhotic patients with at least one siderotic nodule detected on previous imaging underwent both computed tomography and magnetic resonance imaging  at 3.0 Tesla. Imaging data was independently analyzed by two radiologists. Siderotic nodule count was determined for each modality and imaging sequence. For each magnetic resonance imaging technique, siderotic nodule conspicuity was assessed on a 3 point scale . More nodules were detected by susceptibility weighted imaging (n = 2935) than any other technique, and significantly more than by T2* weighted imaging . Lesion conspicuity was also highest with susceptibility-weighted imaging, with all nodules found to be moderate (n = 6) or strong (n = 40); a statistically significant difference (p<0.001).Susceptibility-weighted imaging had the greatest lesion conspicuity and detected the highest number of siderotic nodules suggesting it is the most sensitive imaging technique to detect siderotic nodules in cirrhotic patients. Iron is a critical element for human life, and the majority of human iron exists in heme-based proteins (predominantly hemoglobin) or ferritin, an iron storage protein. The liver is the primary organ for iron storage and metabolism. Numerous human diseases are associated with abnormalities of iron metabolism, and in the liver, the pathogenesis of many hepatic diseases result in the accumulation of excess hepatic iron. Excess hepatic iron can result from genetic predisposition (hemochromatosis) or from excess circulating iron, which is typically due to excess breakdown of hemoglobin (hemosiderosis). Many techniques have been developed to detect and quantify hepatic iron content because of its importance in the diagnosis and prognosis of related diseases and for monitoring and evaluating treatment response. The quantification of iron content is most commonly indirectly estimated by serum ferritin level and directly determined by liver biopsy. However, ferritin measurements often do not accurately reflect hepatic iron stores Hepatic nodules can be detected with ultrasound, computed tomography (CT), or magnetic resonance (MR) imaging. Ultrasound lacks the ability to discriminate iron content, and the sensitivity of computed tomography for the detection of iron is also limited Hepatic cirrhosis is the progressive destruction of normal hepatic architecture with fibrosis and replacement by a range of partially restorative nodules, varying from benign regenerative nodules and premalignant dysplastic nodules to hepatocellular carcinoma. Even in the absence of systemic iron storage diseases such as hemochromatosis, iron can accumulate within regenerative or dysplastic nodules in the cirrhotic liver; these nodules are termed “siderotic nodules” (SN) T2*-weighted imaging is currently the most sensitive MRI technique to detect siderotic nodules in the liver with a reported 80% sensitivity and 95% specificity All research procedures were approved by the Institutional Review Board of the Third Medical Military University and were conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained for all patients.Using an institutional database, a total of 362 patients from December 2009 to December 2010 with clinically diagnosed cirrhosis were identified. From this set of identified patients, 46 patients  agreed to participate in our study and have a specialized set of CT and MR examinations. Patient inclusion was contingent upon meeting the following criteria: 1) ability to sustain a 20 second breath hold; 2) no evidence of iron overload ; 3) at least one siderotic nodule previously detected by CT or MR; and 4) no other lesions, such as cholangiocarcinoma, detected by prior MR or CT; however, patients with hepatocellular carcinoma were included in the study. Of the 46 patients, causes of cirrhosis were hepatitis B , hepatitis C , autoimmune cirrhosis , alcohol abuse , and primary biliary cirrhosis .All subjects were imaged with dual-source CT . Imaging parameters were: 250 mAs, 120 kVp, 1.2 mm beam collimation with a 0.5 s gantry rotation time. Field of view (FOV) was 35 cm, with a reconstruction thickness and interval both of 5 mm. No oral contrast was administered. The examination consisted of noncontrast images followed by three dynamic images acquired 35 s , 70 s , and 180 s (delayed phase) acquired following the intravenous administration of 100–120 ml Ultravist 370  at a rate of 3–4 ml/s.2 and matrix was 320–384 × 250; slice thickness was 5 mm with a gap of 1 mm. Depending upon the liver size, two to three breath holds of 15 to 20 s duration were used for each sequence.MR imaging for all subjects was performed on a 3.0 T whole-body system  using a standard 12-channel matrix coil, without intravenous contrast enhancement. The following MR pulse sequences were used for all patients: transverse T1-weighted 2D gradient echo (GRE) , transverse T2-weighted 2D fast spin echo , transverse T2*-weighted 2D GRE  and transverse abdominal 2D SWI . For all sequences, FOV was 280×285 mmFor abdominal SWI, a recently developed multislice 2D GRE sequence with SWI reconstruction  was used. An SWI dataset consisted of 3 contiguous 10-slice transverse acquisitions through the liver, with the duration of each acquisition suitable for a single breath-hold (less than 20 s).0 inhomogeneity, both of which are increased in abdominal imaging compared to brain imaging. First, complex images from each of the 12 channels were acquired individually and high-pass filtered with a 32×32 filter to reduce artifacts related to both coil sensitivity and magnetic field inhomogeneity. High-pass filtering was achieved by the following steps: 1) Fourier transformation of the complex image to k-space; 2) while keeping the center of k-space (size 32×32) unchanged, the remainder of the k-space matrix was zero-filled; 3) inverse Fourier transform of the zero-filled k-space to obtain a complex low-pass filter, and division of the original complex image with this low-pass filter to remove low spatial frequency components. Then, the high-pass filtered complex images from each channel were weighted by the coil sensitivity factor and combined to generate a single complex image, as described by the adaptive combine method SWI postprocessing was performed automatically on the magnet. Postprocessing of abdominal SWI images was designed to reduce motion artifacts from breathing and cusp artifact  from BNodules that were hyperattenuating compared to background parenchyma were considered to represent siderotic nodules on CT, and nodules that were hypointense to background parenchyma were considered to represent SN on MRI After the combined representative dataset was created for each patient, two readers independently reviewed all 92  examinations on a commercially available imaging workstation . Readers were blinded to previous clinical MR interpretations, pathologic results, and sequences used. For each of the representative slices, the number of SN was determined and the distribution classified as focal (<5 SN per slice), scattered (5–20 SN per slice), or diffuse (>20 SN per slice). For any questionable nodule, consensus review with a third reader was used to decide. Any nodule detected on CT with an attenuation value ≥100 Hounsfield units was considered to represent a calcified lesion and was excluded for SN analysis t-test. Cohen’s kappa method was used to assess inter-reader agreement with respect to diagnosis determined by each reader on the basis of images of different imaging techniques. The reported Cohen Κ is the average Κ values computed for both readers over the entire set of all images.All statistics were computed using dedicated statistical software . P values of <0.05 were considered statistically significant. The number, distribution, and conspicuity of SN were compared by paired The comparison of SN detected by CT, T1-, T2-, T2*-weighted and SWI imaging is illustrated in Overall conspicuity of SN was categorized for each MR imaging technique . Lesion Inter-reader correlation for conspicuity, determined using the Κ coefficient, was estimated at 0.82 based on results provided by each individual reader.Our results of this study evaluating 46 patients with hepatic cirrhosis and siderotic nodules demonstrated that abdominal SWI detects more nodules than other tested sequences; in particular, almost six times as many patients . While the association between grades of siderotic dysplastic nodules and malignancy has not been established, siderotic dysplastic nodules are thought to represent premalignant lesions We are aware of several limitations in this study. While T2* weighted imaging is the most common clinically used method to detect siderotic nodules, the gold standard for siderotic nodule detection remains histologic; pathological correlation with explant livers would be the ideal reference; lack of pathologic correlation potentially biases results to favor T2* and SWI over other imaging techniques. We also considered all hypointense lesions on T2* and SWI to be siderotic nodules; while this is most likely given our patient subset, other lesions can also appear hypointense. Only histologic explant liver evaluation could definitively exclude all possible nonsiderotic hypointense nodules. The inclusion of patients only with known siderotic nodules introduces a bias, and our evaluation cannot conclusively prove whether SWI would detect siderotic nodules in instances where other MRI imaging methods would detect none; T2* weighted MRI detected at least 5 or more siderotic nodules in all patients. Since only a single representative image from each examination was chosen for evaluation, the potential for undersampling bias exists; evaluation of the entire hepatic volume would have eliminated this possible bias. The use of multiple breath-hold imaging acquisitions to achieve coverage of the entire liver may be difficult or unattainable in some cirrhotic patients, such as those with ascites or reduced pulmonary function. Gastrointestinal air is a known source of magnetic susceptibility and can impart artifacts, and can limit evaluation of some liver regions depending upon anatomic considerations; however, we anticipated that further technological improvements in MR sequence design can minimize such artifacts.In conclusion, we show in this study that imaging using SWI substantially improves both the conspicuity of siderotic nodules and number of detected nodules relative to other techniques, including T2*-weighted gradient-echo imaging; additionally, these results are repeatable as inter-reader agreement is highly correlated. SWI should be a useful addition to current imaging techniques, and may prove to be the most sensitive technique for the detection and characterization of hepatic siderotic nodules in the future.


The challenge in extracting genome-wide chromatin features from limiting clinical samples poses a significant hurdle in identification of regulatory marks that impact the physiological or pathological state. Current methods that identify nuclease accessible chromatin are reliant on large amounts of purified nuclei as starting material. This complicates analysis of trace clinical tissue samples that are often stored frozen. We have developed an alternative nuclease based procedure to bypass nuclear preparation to interrogate nuclease accessible regions in frozen tissue samples.Here we introduce a novel technique that specifically identifies Tissue Accessible Chromatin (TACh). The TACh method uses pulverized frozen tissue as starting material and employs one of the two robust endonucleases, Benzonase or Cyansase, which are fully active under a range of stringent conditions such as high levels of detergent and DTT. As a proof of principle we applied TACh to frozen mouse liver tissue. Combined with massive parallel sequencing TACh identifies accessible regions that are associated with euchromatic features and accessibility at transcriptional start sites correlates positively with levels of gene transcription. Accessible chromatin identified by TACh overlaps to a large extend with accessible chromatin identified by DNase I using nuclei purified from freshly isolated liver tissue as starting material. The similarities are most pronounced at highly accessible regions, whereas identification of less accessible regions tends to be more divergence between nucleases. Interestingly, we show that some of the differences between DNase I and Benzonase relate to their intrinsic sequence biases and accordingly accessibility of CpG islands is probed more efficiently using TACh.The TACh methodology identifies accessible chromatin derived from frozen tissue samples. We propose that this simple, robust approach can be applied across a broad range of clinically relevant samples to allow demarcation of regulatory elements of considerable prognostic significance. The coupling of next-generation sequencing methodologies with classical enzymatic chromatin digestion approaches (eg. DNase I and MNase) has provided global, high-resolution information on chromatin features such as nucleosome positioning and chromatin accessibility ,2. DNasein situ NP40 lysis leaving adherent nuclei also allows DNase I treatment of cells grown as a monolayer [The use of DNase I as a probe of chromatin accessibility was initially described three decades ago ,8, whereonolayer .Serratia marcescens, is composed of two identical subunits of 30 kDa, requires divalent cations for full activity and is generally used to clear cellular protein extracts of DNA and RNA prior to downstream analysis [In order to define accessible chromatin compartments in samples derived from frozen tissue, where cell numbers are unknown and nuclear preparation is problematic, we hypothesized that digestion of chromatin with more robust nucleases that function in a coarse environment as well as, over broad concentration ranges, might enable the determination of chromatin accessibility in tissue samples. Benzonase, a recombinant endonuclease derived from analysis . It digeanalysis . CyanaseAccessible chromatin has traditionally been identified by DNase I digestion of chromatin using nuclei as starting material  grown in suspension were isolated, resuspended hypotonic buffer and incubated with increasing concentrations of Benzonase and Cyanase. Accessible regions at the c-myc promoter were compared using indirect-end labeling and Southern blotting as previously described . We showTo test whether Benzonase and Cyanase can interrogate nuclease accessible regions in chromatin from frozen tissue, whole livers from C57BL/6 mice were isolated and frozen immediately in liquid nitrogen. We initially compared different procedures to prepare frozen tissues amenable for nuclease treatment without disrupting chromatin integrity. We found that rapid pulverization of frozen tissue into a fine powder prior to digestion results in the best signal to noise ratio (data not shown). Pulverization was performed on dry ice with equipment pre-cooled in liquid nitrogen and pulverized tissue was stored as aliquots Figure  and acceTo verify that the TACh procedure identifies accessible regions associated with regulatory elements of gene expression, we mapped the distribution of hotspots in distal upstream regions, promoters, introns, exons and downstream regions, and correlated hotspot intensity with previously mapped histone modifications  and nuclTranscriptional start sites (TSS) of active genes are occupied by highly mobile nucleosomes  and are To validate that accessible regions identified by the TACh are indeed bona fide nuclease hypersensitive sites, we mapped DNase I accessible regions using nuclei purified from fresh liver tissue Figure A. Using The variation observed between identified hotspots by the nucleases might be explained by the intrinsic methodological differences between TACh and the DNase I-based assays. Specifically, TACh is performed in intact cells with minimum manipulation prior to digestion, while the DNase I assay is performed on nuclei that take at least an hour to process. Alternatively, differences between DNase I, Benzonase and Cyanase can be a consequence of sequence specificity for DNA recognition and cleavage by each of the endonucleases. Benzonase preferentially digests dsDNA enriched for Gs and Cs  while DNNuclease accessible sites are maintained by the targeted recruitment of remodeling complexes by transcription factors . The traIdentification of nuclease accessible sites is a powerful approach to annotate regulatory regions of the genome. In cell lines and fresh tissue where nuclei can be isolated with high efficiency, DNase I has been used for decades to probe for accessibility. However DNase I is inhibited by actin and DNase I can therefore not be used on frozen tissue samples where nuclei cannot be purified in sufficient amounts while maintaining nuclear integrity. Here, we show the use of two other nucleases, Benzonase and Cyanase, in probing chromatin accessibility in frozen whole tissue samples. The digestion patterns of chromatin with Benzonase and Cyanase are remarkably similar and the identified accessible regions correlate with low nucleosomal occupancy, epigenetic marks of euchromatin and levels of transcription of proximal genes, validating the use of TACh for identification of regulatory elements in the genome. Comparisons with DNase I accessibility in liver tissue display a significant overlap between DNase I accessible sites and accessible regions identified by TACh. The similarities are most pronounced at highly accessible regions, whereas identification of less accessible regions tends to be more divergent between nucleases. Interestingly, we show that some of the differences between DNase I and Benzonase relate to their intrinsic sequence biases, with Benzonase and Cyanase preferring GC-rich sequences and disfavoring AT-rich sequences.Currently, formaldehyde-assisted isolation of regulatory elements (FAIRE) is the only reported method to identify regulatory elements in frozen tissue samples ; howeverFor clinical samples, TACh provides several advantages compared to current nuclease based methods. 1) TACh uses frozen tissues, which eliminates the need for immediate processing of freshly acquired tissue and is, therefore, compatible with the acquisition of samples under routine clinical conditions. 2) The ability to store whole tissue, fragments or pulverized powders provides flexibility and the powder ensures easy generation of matched aliquots that can be used for additional genomic experiments. 3) The ability to interrupt the procedure at multiple steps makes the whole process more amenable to clinical situations and laboratories. 4) The quick processing and the use of whole cells are likely to minimize nuclear damage and the loss of dissociated chromatin components. 5) Because the chromatin structure is preserved, it is likely that the same pulverized tissue can serve as the starting material for ChIP-Seq interrogations of the same tissue.Thus TACh will make it possible to mine the accessible genomes from banks of frozen specimens, including biopsies and resected material from diseased and healthy human tissue, and is likely to assist in understanding the pathophysiology of a number of disease states. In particular, since TACh efficiently identifies accessible CpG islands, the method may be used to characterize chromatin structure rearrangements during progression of cancer, which are often associated with abnormal DNA methylation at CpG island rich promoters, leading to deregulation of numerous genes . IdentifThe described TACh methodology is a robust method for highly sensitive and comprehensive detection of accessible chromatin in samples derived from frozen tissue. The robustness and quick processing time of the assay provides feasible analysis of multiple tissue biopsies and we propose that application of TACh on clinically derived tissue material will provide knowledge on changes in chromatin accessability during progression of diseases.HL-60 (ATCC) suspension cells were cultured in RPMI + 10% FBS in CO2 at 37°C.Male C57BL/6 mice, 8-week old purchased from Harlam Sprague Dawley , were maintained according to the NIH guidelines with a 14-h light, 10-h dark cycle and free access to food and water. Mice were killed by decapitation or cervical dislocation and livers were immediately sectioned into 5-10 mm cubes and frozen in liquid nitrogen or processed for nuclei purification. All animal procedures were approved by the Animal Users and Care Committee, NICHD and NCI, NIH.Cells were collected by centrifugation, washed twice in ice cold cellular wash buffer (CWB) ) and resuspended in (40 million cells/ml) hypertonic lysis buffer (HLB) ). Cells were distributed in 500ul aliquots in 1.5 ml tubes and following addition of 500ul of nuclease digestion (ND) buffer  containing a 3-fold dilutions (from 0.125 units/ml to 6U units/ml) of Benzonase  or Cyanase . This was mixed gently and incubated for 3 minutes at 37°C. Reactions were terminated by the addition of EDTA  and SDS . Proteinase K was added to a final concentration of 0.5 mg/ml and incubated overnight at 45°C. DNA was purified by Phenol/Chloroform/isoamyl (PCI) extraction, ethanol precipitated and processed for Southern blotting.Purified nuclease digested DNA was digested with HindIII overnight and then PCI extracted and ethanol precipitated. Precipitated DNA was SacI digested, RNase treated, extracted by PCI and ethanol precipitated. Digested DNA was separated on an agarose gel and transferred to a nylon membrane and incubated with a biotinylated c-myc probe  overnighLivers were rapidly removed from mice and frozen immediately in liquid nitrogen. Frozen liver fragments were pulverized using a stainless steel pulverizer (Bessman), pre-chilled in liquid nitrogen. Seven hundred mg of frozen , pulverized tissue was transferred to a pre-cooled 15 ml tube and suspended in 4 ml HLB ). The suspension was mixed with a 3 cc syringe fitted with a 19 gauge needle and HLB was added to a final volume of 7 ml. The resulting suspension was passed through a 19 gauge needle 5 times, followed by a 22 gauge needle 5 times and finally a 23 gauge 10 times, using a 3 cc syringe. The suspension was distributed in 500ul aliquots in 1.5 ml tubes using a 23 gauge needle and incubated on ice for 5 minutes. 500ul of ND buffer  containing a 2-fold dilutions of Benzonase  or Cyanase  was mixed gently with the 500ul tissue solution and incubated for 3 minutes at 37°C. Reactions were terminated by the addition of a final concentration of 50 mM EDTA and 0.1% SDS. 50ul RNaseA/RNaseT1 (Ambion) was added and the reaction was incubated over night at 40°C. SDS was brought to a final concentration of 0.75% and incubated for 2 hours at 45°C, after which Proteinase K was added  and incubated overnight at 45°C. DNA fragments of 100-500 bp from a chromatin digestion were purified over sucrose gradients [Livers was removed from mice and homogenized in 8 ml/g tissue of low sucrose buffer ) using a Type B dounce. Crude nuclear pellets were washed once in low sucrose buffer and resuspended in 9 volumes of high sucrose buffer ). The nuclear suspension was aliquoted in 2.0 ml tubes and centrifuged at 16,000xg for 30 minutes at 4°C. Nuclear pellets were combined in buffer ) and DNase I digestions were performed with 20 million nuclei as previously described . Sequenced DNA was aligned to the mouse genome (mm9) using Eland or Bowtie . HotspotRNA-seq ; SREBP-2bp, Base pair; ChIP, Chromatin Immunoprecipitation; ChIP-seq, ChIP combined with massive parallel sequencing; DNase-seq, DNase I digestion of chromatin combined with massive parallel sequencing; FAIRE, Formaldehyde assisted identification of regulatory elements; FOXA2, Forkhead box A2; H3K4me1, Histone3 lysin 4 mono-methylation; H3K4me3, Histone3 lysine 4 tri-methylation; H3K27me3, Histone3 lysine 27 tri-methylation; Mnase, Micrococcal nuclease; RNA-seq, Massive parallel sequencing of RNA; SREBP, Sterol Regulatory Element-Binding Protein; TACh, Tissue accessible chromatin; TSS, Transcriptional start site.The authors declare that no competing interests exist.LG carried out the DNase-seq in liver, participated in TACh-seq in liver, analyzed sequencing data and drafted the manuscript. RB developed the TACh method and performed TACh in cell lines and liver tissue, SJ participated in DNase-seq and TACh-seq in liver and helped to draft the manuscript. SB analyzed sequencing data. HJC helped develop the TACh method. YL and GA performed all the mouse work. CO and GLH participated in the study design and GLH helped to the draft of the manuscript. DL designed and coordinated the study and helped draft the manuscript. All authors read and approved the manuscript.Figure S1. Table of aligned sequence tags. Figure S2: Correlation of sequence tag densities from chromatin digested with different concentration of Cyananse. Figure S3: Correlation of sequence tag densities from chromatin digested with Benzonase and Cyanase. Figure S4: Distribution of AT percentage surrounding DNase I, Benzonase and Cyanase hotspots. Click here for file


Salvelinus fontinalis) and bull trout (S. confluentus) as a case study. We found qPCR to be substantially more sensitive than traditional PCR, with a high probability of detection at concentrations as low as 0.5 target copies/µl. We also found that number and placement of base pair mismatches between the Taqman MGB assay and non-target templates was important to target specificity, and that specificity was most influenced by base pair mismatches in the primers, rather than in the probe. We found that insufficient specificity can result in both false positive and false negative results, particularly in the presence of abundant related species. Our results highlight the utility of qPCR as a highly sensitive eDNA tool, and underscore the importance of careful assay design.Environmental DNA (eDNA) is being rapidly adopted as a tool to detect rare animals. Quantitative PCR (qPCR) using probe-based chemistries may represent a particularly powerful tool because of the method’s sensitivity, specificity, and potential to quantify target DNA. However, there has been little work understanding the performance of these assays in the presence of closely related, sympatric taxa. If related species cause any cross-amplification or interference, false positives and negatives may be generated. These errors can be disastrous if false positives lead to overestimate the abundance of an endangered species or if false negatives prevent detection of an invasive species. In this study we test factors that influence the specificity and sensitivity of TaqMan MGB assays using co-occurring, closely related brook trout ( Environmental DNA (eDNA) is DNA extracted from an environmental sample without isolating the target organism Much has been written about the problems associated with nonnative species, from economic to ecological impacts e.g., . NonnatiAlong with its unique advantages, eDNA monitoring presents a unique set of challenges and limitations. For example, typical non-invasive DNA sampling targets a particular species by sampling hair, scat or other tissues deposited by the target organism. Thus, most of the DNA will either be from the target species, or from distantly related species . In eDNA approaches, target organism DNA likely represents a minority of the total DNA collected, and the sample may be dominated by DNA from closely related, non-target species. Although eDNA from aquatic systems can be concentrated via precipitation Stream systems also pose special challenges for eDNA analyses. In lentic systems , equilibrium DNA concentrations are determined primarily by the rate of new DNA entering the water and the rate of DNA leaving the system through degradation t, and it can be compared to standards of known copy number to accurately estimate initial target quantity in the unknown sample Hypophthalmichthys molitrix and H. nobilis) H. molitrix and 207 copies/µL for H. nobilis using 1 µL template in traditional PCR reactions Quantitative PCR (qPCR) using TaqMan™ assays with minor groove binding probes  may be a particularly useful tool for eDNA. TaqMan MGB assays use two primers to amplify a small section of target template. Nested within this amplicon is a binding site for a probe, which is labeled with a fluorescent reporter dye on one end and a quencher molecule on the other. When intact, the quencher absorbs the reporter dye signal via fluorescent resonance energy transfer (FRET). The probe is cleaved by the 5′–3′ nuclease activity of the DNA polymerase during extension, thus separating the quencher and resulting in an increased fluorescent signal. As a result, the level of fluorescence is proportional to the quantity of template. As PCR progresses and quantity of amplicon increases, so does the level of fluorescence, creating an amplification curve where fluorescence increases rapidly early in PCR, but slows to a plateau as reagents are consumed. The greater the initial quantity of target template, the earlier in PCR this curve becomes detectable. The cycle number at which the curve becomes clearly distinguishable from the background fluorescence is called the CTaqMan MGB probes should also be substantially more target-specific than traditional PCR. Minor groove binding probes use a 3′ modification to allow construction of very short probes and are extremely sensitive to base pair mismatches Salvelinus fontinalis) are an invasive fish species that has been widely stocked into small streams, where it commonly displaces native salmonids including bull trout    e.g.,  and cutti) e.g., . To creaHere we develop TaqMan MGB assays for detection of these two closely related species of char. We focus on testing the reliability and sensitivity of an assay for detection of brook trout––both in detecting isolated brook trout DNA and when applied to mixtures of DNA from multiple species.cytochrome b gene (cyt b) which are highly divergent across species of salmonids that often co-occur in the western United States We designed two TaqMan MGB assays for detection of brook trout and one assay for detection of bull trout. These assays target ∼150 base pair sites on the BRK1) was designed to maximize base pair differences between brook trout and non-brook trout salmonids within the probe-binding region (BRK2) and bull trout assay (BUT1) were designed to maximize the base pair differences between target  and non-target salmonids in the primer-binding regions and contain at least one base-pair difference in the probe-binding region , Arctic char  Cottus spp.) cyt b for 10 bull trout, 6 brook trout and 2 lake trout  respectively. Brook trout and bull trout were collected from 10 and 6 streams respectively, distributed across western Montana and northern Idaho . Lake trout were collected from Flathead Lake, Montana by The Confederated Salish and Kootenai Tribes, Division of Fish, Wildlife, Recreation, and Conservation. All brook trout were derived from introduced populations, but the lineages are of unknown origin. Lake trout were stocked into Flathead Lake in the early 20th century from an unknown source . DNA from caudal fin tissue was extracted using the DNeasy Tissue and Blood Kit  according to the manufacturer’s specifications. The bull trout and brook trout cyt b region was amplified using primers Verspoor_F1Jane_R (5′-CACAACTATGAGGACAAGGATCG-3′), while the two lake trout were amplified using the primers L14724 and H15915Verspoor_F1, Verspoor_F2,Jane_F (5′-GATTAACTCCGACGCTGACAA-3′) and Jane_R (above) for bull trout and brook trout, and the primers Lc2, Lc3H15915Because no published BRK1 assay. This region was compared with published sequences for closely related species, including Dolly Varden , lake trout, and Arctic char   to maximize within-probe base pair mismatches. Primers for a region surrounding this probe were then designed by the assay manufacturer (Applied Biosystems) to optimize assay performance. To design the BRK2 and the BUT1 assays, we used sequences generated for brook trout, bull trout, and lake trout (above), as well as published sequences including brook trout, brown trout , Arctic grayling , and rainbow trout (Oncorhynchus mykiss)  to maximize within-primer base pair mismatches. These assays were designed using PrimerExpress v3.0 software (Applied Biosystems). One bull trout that was sequenced was found to differ at a single base pair within the forward primer region of the BUT1, however, this did not show any effect on assay efficiency.We used published brook trout sequence data  to select a probe-binding region for the 2O following fast cycling conditions  on a StepOne Real-time PCR Instrument (Applied Biosystems). Because real-time PCR is highly sensitive, and therefore susceptible to contamination, all experiments were set up inside of an enclosure which was irradiated with UV for 1 h prior to each use, along with all consumables and pipettes. Reagents were also aliquoted into small quantities prior to experiments such that each reagent tube was only opened a single time in a PCR product-free environment.All assays were obtained from Applied Biosystems, and contained a primer set and a FAM-labeled minor groove binding, non-fluorescent quencher (MGB-NFQ) probe . All exphttp://fwp.mt.gov/fishing/mFish/), and Miller Creek , which contains a mixture of brook trout and non-target salmonids . Similar to some previous studies We collected eDNA from two streams in west-central Montana known to contain brook trout: Plant Creek,  which is dominated by brook trout and does not contain any other char species . The brook trout DNA solution was created by digesting brook trout tissue in 180 µl ATL  and 20 µl ProteinaseK (Qiagen), then pouring this solution onto a paper filter which was then extracted using the MoBio Powerwater™ DNA Extraction Kit (above). DNA extracted for sequencing (above) was used for the brook/bull trout mixed solution. These two tissue-derived solutions were diluted 1∶100 in IDTE pH 8.0 prior to quantification.6 copies/µl. This solution was then serially diluted to create known quantity standards to estimate brook trout mtDNA concentration in the four sources of DNA over a series of two PCR plates. Each standard, unknown quantity sample, and a no template control (NTC) were run in triplicate on each plate. We used these qPCR quantification data to estimate brook trout mtDNA copy number concentration for each of the four stock solutions  containing the 139-bp sequence of interest (probe and primers) to create a standard curve. The synthetic gene arrived desiccated and was re-suspended in 200 µl of IDTE pH 8.0. We then linearized 20 µl of this stock using Pvu1 restriction digest (New England BioLabs) and purified this product using the PureLink PCR Micro Kit (Invitrogen – Life Technologies Corporation). The final elution using the PureLink elution buffer was 10 µl, to which we added 10 µl of IDTE pH 8.0 for a final volume of 20 µl. This solution was quantified on a Qubit 2.0 fluorometer (Invitrogen), and diluted in IDTE pH 8.0 to a final stock concentration of 10olutions . Based oWe developed a probability of detection curve from each dilution series by running multiple replicates of each dilution. The brook trout tissue-extracted sample dilution series was run a total of 40 replicates per dilution. The Plant Creek, Miller Creek, and the brook trout-bull trout mixed DNA dilutions were run a total of 26 replicates at the 12.5 target copies/µl dilutions and 32 replicates at the 2.5 and 0.5 target copies/µl dilutions. Each PCR plate included a NTC in triplicate. A replicate was counted as a success if there was amplification above the threshold level of fluorescence within 45 cycles. The probability of detection at each dilution was calculated as the proportion of successes out of the total number of replicates.BRK1 and BRK2, bull trout DNA for BUT1), expressed as “proportional S/N.” The target species has a proportional S/N of 1.0, and in the ideal assay, all non-targets would have a proportional S/N equal to that of a NTC (no amplification).Specificities of the three assays were compared using DNA of target (brook trout or bull trout) and non-target salmonids  as the PCR template. DNA was extracted from a piece of fin tissue from brook trout, bull trout, lake trout, and brown trout  using the Qiagen DNeasy® Tissue and Blood Kit (Qiagen) following the manufacturer’s instructions. Reactions for each DNA template source were run in triplicate to identify outliers. To standardize specificity measures, we measured the signal divided by the noise (S/N), which is endpoint fluorescence  at 40 cycles) divided by fluorescence at a single cycle BRK1 and BRK2 assays, extracted brook trout DNA (target) was diluted into bull trout and lake trout DNA (non-target) solution at ratios of 1∶10 and 1∶100. For the BUT1 assay, extracted bull trout DNA (target) was diluted into brook trout and lake trout DNA (non-target) solution at ratios of 1∶10 and 1∶100. Each of these dilutions was run in triplicate.We used extracted DNA from the assay specificity tests (above) to compare the ability of the three assays to distinguish target DNA in the presence of high concentrations of non-target DNA. For the The standard curves were suitable for approximating copy number of unknown samples  relative to that of known standards to estimate initial template quantity; the lower the Ct value, the greater the estimated copy number. In the presence of bull trout DNA, the Ct values were less than expected, which could result in a substantial overestimate of copy number. There was no apparent effect on amplification efficiency for either brook trout assay when brook trout DNA was mixed into high concentrations of lake trout DNA solution  is rare relative to sympatric, closely related species , designing a target-specific assay is particularly important. We found that assay specificity was most influenced by base pair mismatches in the primers, rather than mismatches in the probe. We also found that the location of these mismatches was important. In general, as primer base pair mismatches increased, fluorescence of non-target templates decreased  and non-BRK1 , which hBRK1 . This prsolation  and 3b.location in the primers also influenced our results. The BUT1 assay was strongly affected in mixed bull/lake trout samples, even though there are more base pair mismatches between the BUT1 assay and lake trout (5) than between BRK1 and lake trout (3), which was unaffected by brook/lake trout mixtures. Base pair mismatches near the 3′ end of the primer have a much larger impact on specificity than those in other regions, and 5′ mismatches generally have very little effect on specificity  has been successful t values, which produces inaccurate DNA quantity estimates and could lead to an inaccurate estimate of fish abundance (e.g., Environmental DNA detection may be especially useful in situations where the target species is too rare to be identified using alternative approaches, or where alternative sampling approaches are unreasonably expensive or labor-intensive. However, these are also the circumstances where it is most important to understand the likelihood of false positives and false negatives. False negatives can occur because the quantity of target species eDNA collected falls below a detection threshold or because non-target substances interfere with the assay. Pumping large water samples and capitalizing on the extreme sensitivity of qPCR minimizes the risk of false negatives, as would setting the sampling scheme in a formal occupancy estimation framework e.g., . Howeverce e.g., . Furtherce e.g., . There iHybridization poses an additional consideration when using these assays. Brook trout and bull trout are able to hybridize The hybridization issue illustrates the more general problem that target DNA presence may not always indicate target species presence. Besides hybridization, false positives may result from the extreme sensitivity of eDNA monitoring. Even when diluting to an average of two target copies/reaction, we had better than 70% amplification success with stream-derived DNA samples. This represents such a small quantity of template that detected DNA could derive from some source other than a live organism. Contamination by researchers in the field or lab is an obvious issue, but there are other possibilities as well. For example, the feces of piscivorous birds or mammals may contain prey DNA


Aspergillus nidulans asexual differentiation is induced by Upstream Developmental Activators (UDAs) that include the bZIP-type Transcription Factor (TF) FlbB. A 2D-PAGE/MS-MS-coupled screen for proteins differentially expressed in the presence and absence of FlbB identified 18 candidates. Most candidates belong to GO term classes involved in osmotic and/or oxidative stress response. Among these, we focused on GmcA, a putative glucose-methanol-choline oxidoreductase which is upregulated in a ΔflbB background. GmcA is not required for growth since no differences were detected in the radial extension upon deletion of gmcA. However, its activity is required to induce conidiation under specific culture conditions. A ΔgmcA strain conidiates profusely under acid conditions but displays a characteristic fluffy aconidial phenotype in alkaline medium. The absence of asexual development in a ΔgmcA strain can be suppressed, on one hand, using high concentrations of non-fermentable carbon sources like glycerol, and on the other hand, when the cMyb-type UDA TF flbD is overexpressed. Overall, the results obtained in this work support a role for GmcA at early stages of conidiophore initiation. Aspergillus nidulans is a widely used model organism for industrially or medically important filamentous fungi as well as for the study of basic developmental processes in eukaryotes Aspergillus has been exploited for more than six decades to explore fungal genetics and cell biology A. nidulans starts with the germination of spores, forming vegetative hyphae that extend apically through the deposition of new material at the tip brlA expression. brlA is the master gene for the production of asexual reproductive structures called conidiophores . Since UDA genes are expressed during vegetative phase and all evidence indicates that they play a role at this stage in the signaling leading to conidiation flbB strain protein extract  while 15 had a lower intensity (VWT/VflbBΔ higher than 1.2). Proteins in those spots were identified by mass spectroscopy with the sole exception of spot-16 , reduced levels of the NADP+-dependent glycerol dehydrogenase (spot-14), involved in glycerol and arabinose metabolism, and of three enzymes that catalyze different reactions from pentose metabolism . Finally, spots 9, 10 and 11 corresponded to different isoforms of citrate synthase and spots 7 and 18 were related to enzymes involved in amino acid metabolism.GO term classification for each candidate is shown in Among candidates directly or indirectly repressed through FlbB regulatory activity, spots 1 and 5 corresponded to enzymes from galactose metabolism and the pentose cycle, respectively, while spots 2 and 3 were identified as a pyruvate decarboxylase. Spot-6 is a NADP-specific glutamate dehydrogenase involved in the synthesis of glutamine-derived amino acids. These results suggest that the presence of FlbB in the cell has an influence in the pattern of carbon metabolism during vegetative growth, from glycolytic to gluconeogenic pathways. This influence extends to amino acid biosynthesis. The above mentioned proteins detected in our assay quantitatively represent major metabolic processes, rather than those elements which are expressed in small amounts or are restricted to specific compartments. For example, we could not detect FlbD among the above proteins, despite the fact that this TF has been proven to be directly regulated by FlbB Among the proteins discovered in this screen, we detected one with hitherto unreported importance in the induction of conidiation. The product of gene An8547 (corresponding to spot-4) predictably encodes a glucose-methanol-choline oxidoreductase (hereafter referred to as GmcA) with a proposed role in stress response 2, acts as an electron acceptor and the reaction generates H2O2 as a product Aspergillus nidulans2O2 have widely been linked to development in fungi An8547/gmcA in A. nidulans conidiophore development. Firstly, we confirmed by Northern-blot experiments that An8547/gmcA expression is dependent on FlbB function, and that of its interacting UDA partner FlbE, since both act in a concerted manner gmcA transcript levels are low in vegetative hyphae, increase six hours after the induction of conidiation and return to basal levels again as conidiophore development proceeds . The cDNA sequence was deposited at the GenBank database with the accession number JN872213.Prior to evolutionary and domain architecture analyses, we confirmed that the −40 and 7.1−65, respectively  is highly variable, the  domains . After a domains . The ali domains . Six ortin silico predictions are supported by the observation of GmcA::GFP chimera dispersed throughout the hypha, without any visible accumulation in defined cellular regions or compartments, and not excluded from nuclei (not shown). The same distribution was exhibited in media with different nitrogen sources and during the formation of conidiophores. We predict an intracellular role for GmcA because a HA3x-tagged version was not detected in the culture medium.GmcA is not predicted by SignalP gmcA, ΔgmcA, and the double null ΔflbB;ΔgmcA. When cultured in standard minimal medium (MMA), containing glucose and nitrate as main carbon and nitrogen sources respectively, we observed that GmcA is not required for radial growth  with respect to GmcA, did not result in a loss of conidiation capability (not shown). This aconidial phenotype of ΔgmcA mutants is different in these conditions to that exhibited by a null flbB strain and such fluffy phenotype is not altered in a ΔflbB;ΔgmcA double null strain  strains under inducing conditions in submerged cultures. Spores of all strains were initially inoculated and cultured in fully supplemented liquid minimal medium (MMA) and, then, transferred to carbon or nitrogen starvation media, that is lacking either glucose or nitrate  supplemented with 80mM nitrate was used in these experiments to achieve an extreme ΔgmcA aconidial phenotype (see next section). We found that conidiation of the ΔgmcA strain was rescued when in contact with a wild-type strain, similarly to what was determined for ΔfluG or ΔflbB mutants .The presence in the medium of certain extracellular metabolites precedes the process of asexual development and these compounds act in trans when produced by different fungal strains gmcA strain in contact with either -flbB or ΔfluG mutants . Thus, these results suggest that GmcA either does not act in the synthesis of the signaling molecules controlled by the abovementioned genes or it is linked with these synthesis pathways through non-hierarchical relations.In view of these results, we also tested a ΔgmcA strain in solid media of varying compositions , we observed no phenotypic changes, and concluded that the role of GmcA in the induction of conidiation is not VeA-dependent (not shown). However, we wanted to study the effect of increasing concentrations of different compounds by using glucose as fermentable and glycerol as non-fermentable carbon sources. For a more detailed analysis, carbon sources were added in order to render equimolar carbon concentrations. Nitrate was used again as the nitrogen source instead of urea and the pH of the medium was confirmed to be 8.0±0.5 at the incubation times shown in gmcA fluffy phenotype was not altered  or solid  MMA supplemented with 80 mM nitrate and varying concentrations of glucose or glycerol. brlAα/β expressions in the ΔgmcA strain when 10% glycerol was used but brlA transcripts were not detected in 0.5 or 5% glucose and 1% glycerol. These results explained the phenotypes shown in The suppression of the ΔbrlAα/β and gmcA at the same time points but, in this case, total RNA samples were extracted from mycelia of wild-type, ΔflbB, ΔgmcA and double null strains grown in media containing either nitrate (80 mM) or ammonium (10 mM) as the main nitrogen source and glucose (1%) as the carbon source  medium. Expressions of gmcA, flbB and flbD were then analyzed in wild type, ΔflbB, ΔflbD and ΔgmcA genetic backgrounds . Repression of palcA was achieved using yeast extract (5 g.l−1) and glucose (1%). Phenotypes of recombinant and recipient strains were analyzed under alcA-induced and repressing conditions in either ammonium (10 mM) or nitrate (80 mM) solid minimal medium  were used with the appropriate supplements. Nutrient depletion experiments were performed essentially as described previously 6 spores/ml of each strain under analysis were inoculated in liquid MMA and cultured for 18 hours at 37°C and 250 rpm. Mycelia were then filtered and re-inoculated in fully supplemented MMA, MMA without any carbon source or MMA without a nitrogen source. Images were taken after 20 hours of culture in starved media.Strains and oligonucleotides used in this study are listed in Ammonium or nitrate was added at 10 or 80 mM. The standard concentration of all carbon sources was 1% (w/v). Saline, osmotic or oxidative stress conditions were induced by adding sodium dihydrogen phosphate (0.5 M), sucrose (1 M), hydrogen peroxide (6 mM) or menadione (10–60 µM) to nitrate or ammonium-containing MMA. Induction of asexual and sexual development and localization analyses were carried out as previously described gmcA::gfp overexpression plasmid, a genomic DNA fragment containing the gmcA::gfp fusion was amplified by PCR from strain BD438 using oligonucleotides flbF-gfpFP-alcA and alcA-gmcA-Up. These oligonucleotides contained EcoRI and BamHI restriction sites, respectively, and allowed the ligation with an EcoRI-BamHI digested pALC-pyroA* plasmid flbB or flbD overexpression were obtained previously by Garzia and coworkers alcA promoter were performed in MMA containing 5 g.l−1 yeast extract and 100 mM threonine as carbon source (MMT) essentially as described in 6 spores.ml−1 and their culture for 18 hours at 37°C and 250rpm in MMA and subsequent transfer to MMT for 6 hours.For the generation of the brlA expression in media with different concentrations of either glucose or glycerol were obtained as follows: 106 spores.ml−1 of strains TN02A3 or BD429 were cultured for 18 hours in liquid MMA with 80 mM nitrate and either 1% glucose or 1% glycerol. Mycelia were filtered to induce asexual development and deposited onto solid MMA plates containing 80 mM nitrate and 0.5% or 5% glucose, and 1% or 10% glycerol, respectively. Samples were processed for RNA extraction after 6, 12 and 24 hours of culture at 37°C.RNA samples for the analysis of gmcA, flbB and flbD were verified through Northern blotting.TN02A3, BD177, BD143 and BD429 were used as recipient strains for deletion, tagging and overexpression of the genes of interest. All transformation cassettes for deletion and tagging were generated using the fusion PCR technique gmcA ORF plus 1.5 kb from both the upstream and 3′UTR regions (oligonucleotides gmcA-PP1 and gmcA-GSP4). The cassette was inserted between the two NotI sites of pGEM-T-easy vector (Promega). This fragment was released from the pGEM-T-easy vector after digestion with NotI and inserted into a previously generated pBS  plasmid bearing a truncated pyroA* allele (unpublished), also digested with NotI. We transformed strain BD429 with this recombinant and the empty plasmids and selected single-copy plasmid integrations at the pyroA locus through Southern blotting using specific radiolabelled probes.Strain (BD605) was generated as follows: We amplified a PCR cassette including the For extracellular complementation experiments MCA supplemented with nitrate (80 mM) was used. Strains were point-inoculated onto the solid medium at a separation of 2 cm. After 5 days of cultivation, the contact zone was examined and photographed under a binocular Nikon SMZ800 microscope.To determine spore production in each condition and strain, colony diameter was measured after the incubation time. Spores were collected in 1 ml of an aqueous solution containing 0.02% tween 20 (Acros Organics) and counted. Spore concentration (spores per square centimeter) was determined by dividing spore number with colony area.http://rsb.info.nih.gov/ij/) software.Observation of fluorescent chimeras was carried out using a DMI6000B Leica microscope, equipped with a 63x Plan Apo 1.4 N.A oil immersion lens (Leica), illuminated with a 100w mercury lamp and fitted with a GFP  filter. Images were recorded with an ORCA-ER digital camera (Hamamatsu Photonics) and processed with Metamorph  or ImageJ 1.37  or rabbit anti-hexokinase  monoclonal antibody cocktails. Peroxidase conjugated anti-rat  or anti-rabbit  IgG immunoglobin were used as secondary antibodies. Peroxidase activity was detected with SuperSignal® West Pico Chemiluminiscent Substrate (Thermo Scientific).Samples containing 200 µg of protein were precipitated using methanol/chloroform protocol Protein bands were processed automatically in a Proteineer DP  as described by The isolation of genomic DNA and total RNA as well as the preparation of DNA probes for Southern and Northern blotting were carried out essentially as described previously by P. pulmonarius AAO sequence used in the Plerurotus eryngiiThe 27 sequences of GMC clade 1 as described by −30 and 1−50, respectively, using HMMsearch Retrieval of 500 GMC sequences from the 98 complete Ascomycete sequencing project databases listed on the Hyphal Tip web page  was carried out by scoring for the presence of both GMC Pfam motifs We followed the PHYLIP . Inspection of the model and comparison of QMEAN scores indicated that the cofactor binding domain was a usable model while the SBD was poorly modeled . By mapping the cofactor binding domain of the 3FIM structure onto the GmcA sequence, the amino acids comprising the two domains were identified . This procedure was extended to partition the residues of additional sets of GMCs by editing CLUSTAL alignments with Jalview In order to determine which residues of GmcA comprised each of the two domains, we modeled the sequence onto the PerynAAO structure (PDB ID: 3FIM) using Swiss-Model Workspace Figure S1Phenotype of the ΔgmcA strain reconstituted with an ectopic copy of gmcA integrated at the pyroA locus. Wild-type (TN02A3), ΔgmcA (BD429) and reconstituted (BD605) strains after 60 hours of culture in MMA supplemented with 80 mM nitrate as the main nitrogen source. The ΔgmcA strain shows a marked fluffy phenotype in these conditions (see (TIF)Click here for additional data file.Table S1Genes identified by mass spectroscopy.(DOC)Click here for additional data file.Table S2Compositional characteristics of GMCs and 27 putative Ascomycete GmcA orthologs with BLAST E  = 0.(DOC)Click here for additional data file.Table S3Oligonucleotides used in this study.(DOC)Click here for additional data file.Text S1Characterization of the GmcA sequence and ortholog search.(DOC)Click here for additional data file.


Purpose. To assess for differences in clinical, radiologic, and pathologic outcomes between patients with stage II-III rectal adenocarcinoma treated neoadjuvantly with conventional external beam radiotherapy  or intensity-modulated radiotherapy (IMRT)) versus high-dose-rate endorectal brachytherapy (EBT). Methods. Patients undergoing neoadjuvant EBT received 4 consecutive daily 6.5 Gy fractions without chemotherapy, while those undergoing 3DRT or IMRT received 28 daily 1.8 Gy fractions with concurrent 5-fluorouracil. Data was collected prospectively for 7 EBT patients and retrospectively for 25 historical 3DRT/IMRT controls. Results. Time to surgery was less for EBT compared to 3DRT and IMRT (P < 0.001). There was a trend towards higher rate of pathologic CR for EBT (P = 0.06). Rates of margin and lymph node positivity at resection were similar for all groups. Acute toxicity was less for EBT compared to 3DRT and IMRT (P = 0.025). Overall and progression-free survival were noninferior for EBT. On MRI, EBT achieved similar complete response rate and reduction in tumor volume as 3DRT and IMRT. Histopathologic comparison showed that EBT resulted in more localized treatment effects and fewer serosal adhesions. Conclusions. EBT offers several practical benefits over conventional radiotherapy techniques and appears to be at least as effective against low rectal cancer as measured by short-term outcomes. Colorectal cancer is the 3rd most common malignancy among both men and women in the United States . ApproxiPreoperative external beam radiation has been shown to increase pathological response rates and reduce the risk of local recurrence , but it At this point, only one group has published data on patients with resectable rectal adenocarcinoma who were treated with EBT. Herein, we analyze the preliminary outcomes obtained with EBT and compare them to patients receiving conventional neoadjuvant chemoradiation (3DRT/IMRT) at our institution. The primary goal of this study, therefore, is to compare radiologic, pathologic, and short-term clinical outcomes between EBT and conventional radiation techniques in the neoadjuvant setting.From 2010–2012, 7 patients with locally advanced low rectal adenocarcinoma  were enrolled in a prospective study of neoadjuvant EBT (NCT01226979) at Johns Hopkins Hospital. Patients were required to meet the following inclusion criteria: greater than 18 years of age, histologically confirmed adenocarcinoma of the rectum, able to undergo local staging by MRI and/or EUS demonstrating a T2N1 or T3N0-1 tumor, and ECOG performance status of 0 or 1. Patients were excluded if they had tumors greater than 12 cm from the anal verge, metastatic disease at time of enrollment, positive inguinal or iliac lymph nodes on MRI, PET, or EUS, concurrent malignancy, bulky tumors that would not allow application of the endorectal probe, or previous pelvic irradiation. For comparison, historical controls were obtained by identifying all patients with stage II-III rectal adenocarcinoma who received conventional neoadjuvant chemoradiation with IMRT or 3DRT at our institution from 2008–2012 and went on to surgical resection. Clinical data for patients treated with EBT was gathered prospectively as part of the trial protocol. To gather data for historical controls, retrospective chart review was performed using the electronic patient record (EPR) system after approval by the Institutional Review Board. For all patients, toxicity was evaluated using the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 4.0.Patients in the EBT group were treated with 4 consecutive daily fractions of 6.5 Gy targeted to the tumor and mesorectal lymph nodes without concurrent chemotherapy. Each fraction was delivered over approximately 15 minutes using a flexible silicone intracavitary applicator  positioned in the rectum using fluoroscopic guidance and a microSelectron high-dose-rate iridium-192 remote afterloading system (Nucletron) as described by Vuong et al. [At the initial assessment for all patients, surgery was preplanned according to the standard of care to take place from 6 to 8 weeks following completion of neoadjuvant therapy. All patients were able to undergo surgical resection after neoadjuvant therapy consisting of total mesorectal excision (TME) accomplished as part of a lower anterior resection (LAR) or abdominoperineal resection (APR) procedure. When possible, LAR was performed in preference to APR so that the anal sphincter and normal evacuative function could be preserved.V = π/6 × A × B × C, where  A,  B, and  C  are the maximum tumor diameters along the x-,  y-, and  z-axes). Changes in tumor volume after neoadjuvant therapy were calculated and tumor response rates were assessed using the sum of the maximum tumor diameters according to RECIST. Contrast enhanced T1 weighted MRI images obtained pre- and postneoadjuvant therapy were used to delineate and measure any abnormal mesorectal lymph nodes as well as any suspicious appearing lymph nodes in the inguinal or iliac chains measuring greater than 1 cm in diameter.MRI images of the pelvis, including high resolution T2 weighted images of the rectum, were acquired in 3 planes both prior to and following neoadjuvant therapy  stained sections were examined microscopically. Final pathologic stage, tumor size, nodal status, metastatic disease, and documentation of treatment effect were recorded. If present, lymphovascular invasion and positive surgical margins were also noted. Tumors considered to be completely responsive to preoperative therapy had no histologic evidence of residual carcinoma. Tumors with microscopic disease or large areas of residual carcinoma were considered partially responsive or nonresponsive to treatment, respectively. Slides from 5 randomly selected patients from each treatment group were rereviewed by a blinded pathologist to evaluate for any differences in radiation-induced treatment effects between the EBT, 3DRT, and IMRT groups.U  test. Comparison of means among three or more groups  was performed using a one-way analysis of variance (ANOVA). An alpha level of less than or equal to 0.05 was considered significant in all cases.Statistical analyses were performed with IBM SPSS Statistics software, version 19 . Patient characteristics consisting of continuous and dichotomous variables were summarized using descriptive statistics. Comparison of proportions between two or more groups was performed using the Pearson chi-squared test. Comparison of means between two groups  was performed using the nonparametric Mann-Whitney  P > 0.05; P = 0.007).All patient characteristics are summarized in P < 0.001). This reduction is not unexpected given the shorter time course of EBT (4 days) compared to 3DRT or IMRT (5-6 weeks). More interestingly, however, the time elapsed from conclusion of neoadjuvant therapy to surgical resection was also reduced for patients who underwent EBT (P = 0.038), despite the fact that all surgeries were similarly planned to take place 6–8 weeks following completion of neoadjuvant therapy. All 3 groups demonstrated similar median reductions in CEA levels after neoadjuvant therapy (P = 0.36). Fewer patients experienced grade 1 or 2 acute toxicity in the EBT group than in the external beam group (P = 0.025). Grade 3 toxicity was rare, occurring in one patient from each of the 3 groups with all 3 incidents taking the form of proctitis. No grade 4 toxicity was reported. Rates of sphincter preservation and postoperative complications were similar among the 3 groups. Given the natural history of rectal adenocarcinoma, length of followup was not sufficient to perform informative analyses of survival and disease progression; however, preliminary results are given here to allow for comparison of EBT to conventional neoadjuvant therapy at the current length of followup. The rates of overall survival and local recurrence free survival at 6 months were 100% in all 3 groups. The rate of distant metastasis at 6 months was 0% for EBT, 7% for 3DRT, and 9% for IMRT. Thus, at a 6-month time point, EBT appears noninferior to conventional neoadjuvant chemoradiation using 3DRT or IMRT.Clinical outcomes of interest included time to surgical resection, change in CEA level after neoadjuvant therapy, acute toxicity, sphincter preservation, and postoperative complications; these outcomes are summarized in n = 7 for EBT, n = 10 for 3DRT, n = 7 for IMRT). All 3 groups showed a striking response to neoadjuvant therapy, with similarly marked reductions in tumor volume. Thus, EBT achieved a comparable degree of reduction in tumor volume as measured on MRI after only 4 days of treatment without chemotherapy as 3DRT and IMRT achieved over 5 to 6 weeks with concurrent 5-FU. Tumor response rates according to RECIST were also similar to the 3 treatment techniques . Rates of margin positivity, lymph node involvement, and lymphovascular invasion were similar among the three treatment groups.Pathologic outcomes of interest included tumor complete response rate, surgical margin status, lymph node involvement, and lymphovascular invasion; these outcomes are summarized in Surgical pathology slides from 5 cases in each of the 3 treatment groups  were randomly selected for rereview by a blinded pathologist to assess for qualitative differences in microscopic treatment effects. The surgical resection specimens showed disruption of tumor architecture (regions of necrosis and the presence of mucinous pools with sparse floating tumor cells) and radiation treatment effects for all 3 radiotherapy techniques. However, the distribution and degree of radiation-induced changes throughout the layers of the rectal wall were different for EBT versus conventional 3DRT/IMRT . Compared to the conventional external beam group, specimens from EBT patients demonstrated more pronounced radiation-induced changes to the superficial layers of the rectal wall  in the region where the tumor was located . The mucIn specimens from patients treated with conventional external beam radiation, pathologic findings were similar in nature, but opposite in distribution with deeper layers of the rectal wall being more prominently affected than more superficial layers . Ulcerat The current standard of care for locally advanced resectable adenocarcinoma of the rectum (AJCC stage II-III) is neoadjuvant chemoradiation with a 5-FU-based regimen, followed by total mesorectal excision and adjuvant FOLFOX chemotherapy . Convent One of the main goals of rectal cancer treatment is sphincter preservation to maintain normal bowel function. Sauer et al. found that neoadjuvant conformal chemoradiation results in increased rates of sphincter preservation; however, long-term studies have demonstrated an overall decline in anorectal function . For thiHigh-dose-rate (HDR) endorectal brachytherapy (EBT) is a possible alternative to conventional external beam radiation. It has been used in various malignancies  to deliver high doses of radiation to the tumor over a short period of time. HDR EBT delivers endoluminal radiation to the mucosal surface overlying the rectal tumor in four fractions of 6.5 Gy  over one week. Its rapid dose fall off limits the exposure of the normal surrounding tissues to radiation , therebyThere is limited data on the clinical outcomes and therapeutic benefits of EBT. Data on high-dose-rate brachytherapy for rectal cancer has only been published from one institution  by Vuong et al., who has treated over 300 patients with 29% achieving a pCR and 37% with only microscopic disease at the time of resection, while less than 1% experience acute grade 3 to 4 toxicities [Radiologic analysis according to RECIST showed similar tumor response rates in EBT, 3DRT, and IMRT patients. Radiologic complete response (rCR) was observed in 1 patient in the EBT and IMRT groups, while no patient in the 3DRT group had an rCR. Interestingly, pathologic complete responses (pCR), defined as absence of any residual tumor cells, occurred at higher frequency than rCR in all 3 groups, with 3 EBT patients, 2 IMRT, and 1 3DRT patient manifesting pCR at surgery. Of the 3 patients who demonstrated pCR in the EBT group, 1 had an rCR, 1 a radiologic partial response (rPR), and 1 had radiologically stable disease (rSD) . This suOne concern in treating rectal tumors with EBT instead of conventional external beam radiation is lack of sterilization of pelvic lymph nodes as a result of the rapid dose falloff associated with EBT, which covers only the mesorectal lymph nodes with little to no coverage of pelvic nodes. Thus, it is conceivable that EBT may lead to higher lymph node metastasis rates and local recurrence. For that reason, patients are selected for EBT based on pathologic lymph node status by imaging. Patients are excluded if positive lymph nodes are identified in the pelvis outside the mesorectum prior to treatment. It is encouraging that in our study preoperative MRI showed no development of pelvic node involvement for any of the 7 patients who received EBT. Furthermore, the one patient in the EBT group with radiologic involvement of mesorectal lymph nodes on pretreatment MRI exhibited complete resolution of nodal involvement on post-EBT imaging. Although limited in their generalizability by the small sample size, these findings suggest that when patients are carefully selected for neoadjuvant EBT , there is a low likelihood that they will develop radiologic evidence of N2 disease prior to surgery. This evidence confirms data presented by Vuong et al. documenting a 5-year local recurrence rate of 5% in N0-N1 patients treated with EBT, which likely indicates a 5% or lower rate of spread to pelvic lymph nodes prior to surgery . HoweverRadiation-induced injury to the rectum is well documented and characteristic histologic changes include architectural disruption and atrophy, goblet cell loss, shortened crypts, a thickened and distorted muscularis, intestinal wall fibrosis, serosal thickening, and vascular sclerosis , 29. A s Our study is the first to describe the histologic differences in treatment effect of EBT compared to conventional external beam radiation seen on pathologic examination of rectal adenocarcinoma resection specimens. In general, the types of histologic changes induced by EBT and conventional external beam radiation were similar, consisting of mucosal ulceration, fibrosis and hyalinization of the lamina propria, degeneration of the muscularis propria, and vessel wall hypertrophy and sclerosis as well as formation of serosal adhesions. Notably, however, the distribution and degree of these changes throughout the layers of the rectal wall were distinct for EBT. TME specimens from patients who received conventional external beam radiation demonstrated moderate radiation-induced changes diffusely throughout the rectal wall. Specimens from patients treated with EBT, on the other hand, displayed these changes along a gradient, with intense treatment effect apparent in the superficial layers of the rectal wall , but progressively reduced treatment effect in each of the deeper layers . These contrasting distributions of treatment effect suggest that EBT may achieve a more potent localized ablative effect on the tumor and immediately surrounding rectal tissue than does conventional external beam radiation, but may not be as effective in sterilizing the serosa.It follows that careful patient selection is critical for successful implementation of EBT. Patients with T1–T3 lesions may derive considerable benefit from the high ablative potential of EBT and would be considered viable candidates because their tumors can be adequately covered with EBT without extreme doses to the rectal wall. If tumors are more than 3-4 cm from the rectal wall, EBT may cause increased proctitis. Our results, as well as the data reported by Vuong et al. , 17–22. Modern staging performed with endorectal ultrasound (EUS) and pelvic MRI has been shown to attain a high degree of accuracy in determining the T stage of rectal tumors. EUS has demonstrated a sensitivity of 90% and specificity of 75% for identifying T3 tumors, while MRI has a sensitivity of 80–86% and specificity of 71–76% –35. In pFinally, the limited range at which radiation treatment effects were observed for EBT on histopathologic examination in our study provides a rationale for the lesser degree of toxicity experienced by patients in the EBT group. Sauer et al. reported a 27% incidence of grade 3 to 4 acute toxicity as a result of neoadjuvant chemoradiation for rectal cancer . More reOur study was primarily limited by a small number of patients and a short period of followup. These limitations precluded definitive survival analysis, but did not hinder evaluation of several clinical, radiologic, and pathologic outcomes of interest. The fact that only data on EBT patients was collected prospectively, while data on 3DRT and IMRT patients was collected retrospectively, introduces the biases inherent in retrospective studies to our analysis. It is also possible, though unlikely, that the difference in gender distribution between the EBT, 3DRT, and IMRT groups could confound our analyses, especially in regard to toxicity considering the different organs at risk in the pelvic region between males and females. Further followup of the EBT patients included in this study, as well as future EBT patients , will be needed to determine median overall survival and thus estimate the impact of EBT on this primary oncologic outcome.Comparison of preliminary EBT trial data to historical controls treated with conventional external beam radiation reveals that patients treated with EBT experience less toxicity and shorter time to surgery without compromising margin or lymph node status at resection. Followup was not sufficient for survival analysis, but EBT appears noninferior to 3DRT and IMRT at 6 months. EBT alone administered over 4 days achieves similar radiologic and favorable pathologic tumor response rates when compared to 5-6 weeks of conventional chemoradiation. EBT showed a more intense local ablative effect on histopathologic examination, suggesting a greater likelihood of achieving pathologic complete response and, consequently, improved long-term outcomes. Furthermore, radiation-induced changes due to EBT were tightly localized to the area of the tumor with greater sparing of normal tissues including small bowel, likely explaining the lower rate of toxicity observed in comparison to 3DRT and IMRT. Careful patient selection using EUS and MRI is necessary to ensure that patients with T4 tumors that extend beyond the range of the radioisotope used for EBT are not offered this therapy. In summary, EBT appears to be a promising mode of neoadjuvant treatment for low lying rectal adenocarcinoma. Longer followup and a larger multicenter study are needed to conclusively evaluate the potential of EBT to produce a survival benefit in this patient population.


This dataset holds great promise as a comprehensive resource for de novo identification of protein-protein interactions or to underpin and prioritize candidate protein interactions from other studies. To demonstrate the potential use of our dataset we focussed on the mitochondrial translation machinery. Our results show that mitoribosomal complexes can be analyzed by blue native gel electrophoresis, as at least four distinct complexes. Analysis of these complexes confirmed that 24 proteins that had previously been reported to co-purify with mitoribosomes indeed co-migrated with subunits of the mitochondrial ribosome. Co-migration of several proteins involved in biogenesis of inner mitochondrial membrane complexes together with mitoribosomal complexes suggested the possibility of co-translational assembly in human cells. Our data also highlighted a putative ribonucleotide complex that potentially contains MRPL10, MRPL12 and MRPL53 together with LRPPRC and SLIRP.Complexome profiling is a novel technique which uses shotgun proteomics to establish protein migration profiles from fractionated blue native electrophoresis gels. Here we present a dataset of blue native electrophoresis migration profiles for 953 proteins by complexome profiling. By analysis of mitochondrial ribosomal complexes we demonstrate its potential to verify putative protein-protein interactions identified by affinity purification – mass spectrometry studies. Protein complexes were extracted in their native state from a HEK293 mitochondrial fraction and separated by blue native gel electrophoresis. Gel lanes were cut into gel slices of even size and analyzed by shotgun proteomics. Subsequently, the acquired protein migration profiles were analyzed for co-migration  Perturbed protein-protein interactions can have strong negative effects on cell viability, which in turn may have devastating effects in an organism. This is exemplified by the severe clinical syndromes that are associated with assembly defects of the mitochondrial oxidative phosphorylation (OXPHOS) complexes via its proteolytic peptides by mass spectrometry in-silico protein migration profiles. The outline of this method is schematically shown in et alBlue native gel electrophoresis (BNE) has been developed to study native protein complexes de novo protein-protein interactions or drive validation of predicted protein-protein interactions from e.g. affinity purification – mass spectrometry (AP-MS) experiments. The latter application is of particular interest as the mass spectrometric identification of co-purified proteins on itself does not provide physicochemical information about the actual interactions themselves or the size of the complexes One of the advantages of complexome profiling over the classical 2D BN SDS-PAGE method is that the electrophoretic profiles are stored in a data matrix of proteins (rows) with gel slices (columns) where each cell contains the respective relative abundance. This allows the unambiguous analysis of protein co-migration by computational methods such as protein correlation profiling via different quantitation methods was previously used to deliver the proof-of-principle for the approach In this paper we present the first complexome profiling dataset from human cells which can be used by researchers to support alleged protein-protein interactions, identify novel protein-protein interactions, or to prioritize candidate protein interactors. A small subset of the protein profiles generated Four of the HCL clusters were of particular interest as they contained proteins of the 28 S and 39 S mitochondrial ribosome (mitoribosome) which has not been analyzed by BNE thus far. Recent publications reported interactions between many different proteins and the mitoribosome based on AP-MS results n-dodecyl b-d-maltoside(approximately 1 gr/gr protein). The suspension was incubated for 10 minutes on ice to solubilize proteins and subsequently centrifuged at 10 000×g for 25 minutes at 4°C. The concentration of solublilized proteins in the supernatant was determined using the MicroBCA protein assay kit (Pierce) prior to addition of BN sample buffer. BN gels (4–12% and 5–15% acryl amide gradient gels) were cast using Bio-Rad mini PROTEAN® II systems in combination with 1.5 mm spacers. Both gels were loaded with 80 µg of protein per lane and run till the dye front reached the end of each respective gel.HEK293 cells grown in DMEM (Biowhitaker) supplemented with 10% (v/v) FCS and 1% (v/v) penicillin/streptomycin were harvested at ∼80% confluency using PBS. Cells were disrupted by pottering in isotonic buffer and a mitochondrial enriched fraction was obtained by centrifugation according to Vogel in-gel tryptic digestion essentially according to Heide in vacuo centrifugation to remove acetonitrile. Subsequently, stop and go elution (STAGE) tips The resulting gel lanes were cut into 24 gel slices at even distance and each gel slice was subjected to in-house at 100 bar with ReproSil-Pur C18AQ 3 µm 140 Å resin (Dr. Maisch) resuspended in methanol. Proteolytical peptides were loaded directly onto the analytical column using 0.5% (v/v) acetic acid at a flow rate of 600 nl/min. A linear gradient of 12–46% acetonitrile (ACN) with 0.5% (v/v) acetic acid as ion pair reagent was used to gradually elute peptides from the column at a flow rate of 300 nl/min. Following each analysis, the column was washed for 10 minutes with 80% ACN at 600 nl/min and conditioned using 0.5% acetic acid for 10 minutes at 600 nl/min. Intermittent blank injections were performed to minimize carry-over effects between samples.Duplicate measurements for each gel slice were performed by nanoflow reversed-phase C18 liquid chromatography (Agilent 1100 series) coupled online to a 7T linear ion trap Fourier-Transform ion cyclotron resonance mass spectrometer  m/z 524) at 300 nl/min and calibrated using caffeine (m/z 195), MRFA (m/z 524) and Ultramark 1621  standards from the MSCAL5 ProteoMass™ LTQ/FT-Hybrid ESI Positive Mode CalMix kit (Sigma Aldrich). The ICR cell precursor scans were performed using a single microscan at 50 000 resolving power (FWHM at m/z 400) on 1E6 ions or 500 ms maximum injection time (whichever came first). Data dependent acquisition of MS/MS spectra by the linear ion trap used a single microscan at normal scan speed. The automatic gain control was set to 1E4 ions for MS/MS with 400 ms maximum injection time. Collision induced dissociation of precursor ions was performed at 27% normalized collision energy for 30 msec and activation Q = 0.250. An isolation width of 3 Th was used to trap and isolate precursor ions for MS/MS experiments. Dynamic exclusion was enabled to minimize re-analysis of precursor ions .The mass spectrometer was operated in positive ion mode and programmed to acquire a single full MS survey scan in the ICR cell with up to four data dependent collision induced dissociation (CID) fragmentation spectra in parallel by the linear ion trap. The mass spectrometer was tuned on MRFA peptide (in-house developed Perl script. Mascot database searches were performed against the Homo sapiens RefSeq (release 44) database Raw mass spectrometry data was converted into Mascot search engine compatible peak lists by ExtractMsn (Thermo Fisher Scientific) and an in-house developed Perl script was used to generate protein abundance profiles from IDEAL-Q output data. p>0.9) and remaining peptide abundance profiles were ranked by descending similarity score. Finally, the protein abundance profiles were generated as the average profile from the top 5 peptide abundance profiles with highest similarity scores. Protein abundance profiles from duplicate gel slice measurements were averaged for each acryl amide gradient. Finally, a core dataset of 953 protein abundance profiles was compiled that consists of proteins that were detected in both acryl amide gradients.Quantitative information was extracted for valid protein identifications as integrated ion current chromatograms of respective peptides by IDEAL-Q software via DAVID Protein identifiers, gene ontology information, and official gene symbols were obtain Hierarchical clustering for all mitochondrial protein abundance profiles was performed by the multiple experiment viewer (MEV) software Co-migration of mitoribosomal proteins with previously reported interactors from affinity purification – mass spectrometry experiments were analyzed by hierarchical clustering. Proteins reported by Rorbach r = 0.991 and r = 0.994, respectively). However, the hydrophobicity distribution of proteins in our dataset shows a slight shift towards more hydrophobic proteins. This might be explained by the relative high abundance of mitochondrial membrane proteins in combination with the protein extraction and electrophoresis methods that were optimized for the analysis of membrane proteins. This is supported by a significant enrichment for proteins with one or more transmembrane domains with respect to the RefSeq Hs database . The distribution of transmembrane domains for proteins in the dataset as well as the RefSeq Hs database is shown in r = 0.993 versus r = 0.927, respectively).A combined total of 118760 MS/MS spectra were mapped to 3981 proteins or 1766 protein identification groups. Proteins were identified at ≤1% FDR with an average of 9 non-redundant peptides and an average summed Mascot ion score of 417 . Minimum Mascot ion score thresholds to achieve 1% protein FDR or better were determined to be 50, 27, 21, 21, 19 and 16 for proteins with coverage of 1, 2, 3, 4, 5 and ≥6 peptides, respectively. Peptides were identified with an average Mascot ion score of 41  and an average precursor ion mass error of 4.85±3.03 ppm. Analysis of the HEK293 mitochondrial fraction by complexome profiling using two acrylamide gradients resulted in a dataset of 953 proteins where migration profiles could be constructed for both the 4–12% and 5–15% acrylamide (AA) gels. Here, the use of two AA gradient gels allows for cross-validation of protein co-migration and helps to distinguish between protein complexes of similar size. Valid protein and peptide identification data is available from p-value  = 5.65E–18).Because a mitochondria enriched fraction was used in this study, the dataset not only contains mitochondrial proteins but also provides migration profiles of proteins that reside in other cellular organelles. Unfortunately, the mitochondrial localization of many proteins is still unknown, and we therefore included information from the MITOP2 database The protein profiles dataset is provided as Out of the 953 proteins, 209 proteins could be assigned to 24 known heteromeric complexes based on protein description or online information resources Some of the mitochondrial proteins in the dataset were expected to form yet unknown protein complexes. Since the protein profiles were stored in a data matrix, the dataset allowed for the use of computer algorithms in the analysis of protein co-migration profiles. We applied hierarchical cluster analysis (HCL) to categorize the dataset for ease of use, similar to the approach of Heide To investigate the rate at which proteins cluster together by mere chance we constructed 10 randomized matrices in which the abundance values were shuffled for each respective AA gradient gel. For each randomized matrix we performed the same hierarchical cluster analysis that was used for the “real” dataset. To determine the rate at which proteins cluster together we focussed on the 16 known mitochondrial complexes described in Four clusters from the HCL analysis were of particular interest as they contained multiple 28 S and 39 S subunits of the mitoribosome which are shown combined in Results from the HCL analysis for mitoribosomal proteins prompted us to look at these complexes in more detail with respect to previously reported interacting proteins. All mitochondrial proteins that were previously reported to interact with the mitoribosome based on affinity purification studies by Rorbach via the HCL analysis of all mitochondrial protein profiles: ERLIN2 and DLST  and in addition amenable to computational analysis. This allows simple comparisons of migration profiles between proteins of interest or the use of computer assisted analyses to identify proteins with similar migration profiles. This approach provides independent supportive evidence for protein-protein interactions suggested by other methods (e.g. bioinformatic or co-purification studies) and may thus be used for validation purposes and to prioritize candidate protein-protein interactions for functional validation studies. In addition, complexome profiling data can be used to identify putative protein-protein interactions from an independent and untargeted experiment. However, depending on the research question and setup of the complexome profiling experiment, additional experiments are likely required to prove protein-protein interactions since co-migration of non-interacting proteins may occur.Here, we used our dataset to analyze co-migration profiles of mitoribosomal proteins and previously described interactors from co-purification studies. By now, several hundred proteins have been suggested to interact with the mitoribosome based on AP-MS studies. These are likely present in multiple complexes which probably include secondary interactions and false positives. As a first step to elucidate these interactions we looked for co-migrating proteins to support alleged interactions. As expected, our analysis showed that the majority of 28 S and 39 S mitoribosomal subunits co-localize in high molecular mass complexes together with previously reported (putative) interactors from AP-MS studies.bona fide interactors or components of the mitoribosome et al reported that both proteins contribute to mitochondrial translation For the 28 S and 39 S complexes, eight and three previously reported interactors from AP-MS studies were found to co-migrate, respectively. Of these, two proteins  are known The 300 kDa ribosomal complex is rather difficult to put into context. First of all, several proteins were found to co-migrate which might be part of other complexes of similar size. Both VDAC proteins as well as PYCR1 and PYCR2 are known to form homomeric complexes of about 300 kDa. Similarly, both HADHA and HADHB are components of the hydroxacyl-CoA dehydrogenase complex for which the determined size corresponds with the α2β2 configuration of the enzyme. Interaction of the CCDC109A protein product with the mitoribosome seems unlikely from a biochemical point of view as it encodes the mitochondrial calcium uniporter c oxidase (COX) subunits. Recent studies demonstrated that LRPPRC and SLIRP interact in a ribonucleoprotein complex involved in posttranscriptional gene expression regulation in mitochondria et al. showed that knockdown of LRPPRC results in an isolated COX deficiency whereas further knockdown of LRPPRC induces a generalized defect of OXPHOS complexes Our analyses highlighted a putative 200 kDa ribonucleotide complex that appears to contain three MRPL subunits together with LRPPRC, SLIRP and cytochrome Our dataset provides the first available analysis of protein complexes in human cells by complexome profiling. The complexome profiling dataset presented in this paper is not exclusively limited to the analysis of mitochondrial protein-protein interactions since a mitochondrial enriched fraction was used. Data for several hundred non-mitochondrial proteins is also available from our dataset that can be used to analyze protein complexes in other cellular compartments. Although this dataset contains a lot of information, future complexome profiling experiments need to be performed in different cell types, tissues, and differential conditions to completely elucidate the composition of mitochondrial protein complexes in humans. Ideally, future complexome profiling datasets should be merged with existing datasets in order to fully understand the complex dynamics of protein-protein interactions. To this end, the fact that protein electrophoretic migration profiles are stored within a data matrix readily facilitates the combination of present and future datasets.via detection of at least four distinct complexes that are composed of mitoribosomal subunits together with potentially novel and previously reported interactors. In addition, co-migration of multiple proteins associated with the biogenesis of inner membrane complexes together with the 39 S and 28 S mitoribosomal complexes is supportive of co-translational assembly in human cells. Our analysis also exposed a putative 200 kDa ribonucleoprotein complex that potentially contains LRPPRC and SLIRP together with three MRPL subunits. Finally, our data highlights TMEM126A as a putative complex I assembly factor based on co-migration with two known CI assembly chaperones and its sequence similarity with the CI assembly factor TMEM126B which is known to interact with the same two CI assembly factors in other cell types.We have generated a collection of BNE profiles for 953 proteins that can be used to identify novel protein-protein interaction and to underpin or prioritize candidate protein-protein interactions. As an example we have used our dataset to analyze mitoribosomal complexes. Our analysis showed for the first time that mitoribosomal complexes can be analyzed by blue native electrophoresis as demonstrated File S1Protein and peptide identification data.(XLSX)Click here for additional data file.File S2Distributions of the physicochemical protein properties of the complexome profiling dataset and RefSeq Hs database.(PDF)Click here for additional data file.File S3Complexome profiling dataset.(XLSX)Click here for additional data file.File S4Overview of complexome profiling data for known complexes in the dataset.(PDF)Click here for additional data file.File S5Hierarchical cluster analysis of protein profiles.(XLSX)Click here for additional data file.File S6MEV hierarchical cluster analysis file.(ZIP)Click here for additional data file.File S7Hierarchical cluster analysis results for the complexome profiling dataset and shuffled matrices.(PDF)Click here for additional data file.