Hox transcript antisense intergenic lncRNA, HOTAIR, that cooperates with the polycomb repressive complex to reprogram chromatin organization. HOTAIR expression is deregulated in a spectrum of cancers and HOTAIR expression correlates with patient survival. Here, we highlight emerging evidence that supports a role for lncRNAs in cancer with implications for the development of novel diagnostics and therapeutics.The therapeutic response and clinical outcome of patients diagnosed with the same cancer type and that receive identical treatment is highly variable to reflect the genetic heterogeneity within tumor cells. Non-coding RNAs (ncRNAs) are recently discovered molecules that regulate eukaryotic gene expression and represent a significant advance towards a better understanding of the mechanisms that govern cellular growth. NcRNAs are essential for the proper regulation of cell proliferation and survival under physiologic conditions and are deregulated in many pathologies, e.g., human cancers. NcRNAs have been associated with cancer diagnosis, staging, treatment response, metastasis and survival and include distinct subtypes, e.g., long ncRNAs (lncRNAs) and microRNAs (miRNAs). LncRNAs have been linked to essential growth-promoting activities and their deregulation contributes to tumor cell survival. A prominent example is the  Despioutcomes . Drug cooutcomes –21.HOTAIR, to correlate lncRNA expression with metastasis, drug resistance and clinical outcome [Numerous mechanisms may explain the molecular basis of drug resistance and treatment failure. Resistance results from genetic variations within the patient's tumor cell. Recently, the dormant state of cancer stem cells and the epithelial-mesenchymal transition have been linked to chemotherapeutic resistance –27. Whil outcome –40.Studies have revealed that protein-coding and non-protein-coding genes are extensively transcribed within the human genome –44. ProtFor nearly four decades, mutation, amplification or deletions within protein-coding oncogenes and/or tumor suppressors was considered the primary cause of tumorigenesis –57. ThisNcRNAs do not encode proteins but studies have documented their role during the steps of tumorigenesis and the development of therapeutic resistance –64. NcRNin trans to epigenetically silence genes at distant loci. LncRNA transcription may open the chromatin structure and permit access to the transcriptional machinery and enhance expression of neighboring protein-coding genes. Similarly, transcription of lncRNAs near protein-coding loci can repress transcription since the presence of the transcriptional machinery on the lncRNA gene locus physically prevents binding to protein-coding genes. The post-transcriptional roles of lncRNAs are more diverse. Since many lncRNAs are complementary to protein-coding genes, lncRNAs may function in mRNA splicing, editing, transport, translation and degradation. Although lncRNAs are best known for modulating transcription, their post-transcriptional influence on mRNA translation is emerging. Recently, HOTAIR was shown to be a post-translational inducer of ubiquitin-mediated proteolysis. HOTAIR associates with certain E3 ubiquitin ligases bearing RNA-binding domains, such as Dzip3 and Mex3b, as well as with their respective ubiquitination substrates, Ataxin-1 and Snurportin-1 [HOTAIR facilitates the ubiquitination of Ataxin-1 by Dzip3 and Snurportin-1 by Mex3b and accelerates their degradation. These studies have uncovered a fascinating and unforeseen role for HOTAIR in protein degradation.LncRNAs are a heterogeneous group of RNAs that regulate expression at the epigenetic, transcriptional or post-transcriptional level –76. LncRportin-1 . HOTAIR The hallmarks of cancer comprise biological capabilities acquired during the multistep development of tumors , 79. TheHox transcript antisense intergenic RNA HOTAIR is expressed from the developmental HOXC locus located on chromosome 12q13.13 and cooperates with the polycomb repressive complex PRC2 [HOX genes [HOTAIR serves as a modular scaffold, assembling a molecular cargo of specific combinations of enzymes that are equipped to regulate target genes [HOTAIR reprograms chromatin organization and in breast cancer cells promotes metastatic potential [The lex PRC2 . This 2.OX genes , 85. Altet genes . Furtherotential .HOTAIR expression has been investigated in tumor samples from many different cancer types. Healthy and cancer tissue was compared to demonstrate that HOTAIR is deregulated in many tumor types  and liver metastases was shown to correlate with a poor prognosis [HOTAIR correlated with the presence of liver metastases. MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) is another lncRNA that has been associated with metastasis and poor prognosis for patients with NSCLC [MALAT1 resides on chromosome 11q13.1 which has been found to harbor chromosomal translocation breakpoints linked to cancer [in vitro studies, have implicated MALAT1 in the regulation of the invasive potential of cancer cells, in cervical [s Figure . HOTAIR rognosis . In addith NSCLC . MALAT1 o cancer , 92. Othcervical  and lungcervical .CUDR) is a 2.2kb RNA transcript that antagonizes the apoptotic effect of cisplatin in bladder cancer cells [CUDR expression correlates with tumorigenesis as well as cellular growth [CUDR has been shown to render resistance to doxorubicin and etoposide, commonly used for squamous cell cancer. The growth arrest-specific 5 lncRNA Gas5 contributes to glucocorticoid resistance [Gas5 exerts its effects by assuming a secondary structure that is similar to a sequence within the steroid-responsive gene promoter. The result is that Gas5 competitively inhibits steroid receptor binding to the steroid-responsive region of the promoter to prevent transcriptional activation. A role as a tumor suppressor in renal cell carcinoma has also recently been reported [PANDA is a lncRNA increased in a subset of breast cancer cells that contributes to anthracycline resistance, a crucial component of breast cancer chemotherapy [PANDA acts by interacting with the nuclear transcription factor NF-YA to decrease the apoptotic effect. PANDA-depleted fibroblasts have been shown to display increased sensitivity to chemotherapy [H19 knockdown by transfection with antisense H19 oligonucleotides suppresses the multi-drug resistance gene and promotes doxorubicin sensitivity [HULC is a 1.6 kb lncRNA that may act as an endogenous sponge to reduce miRNA levels and inhibit their functional activity [The lncRNA cancer upregulated drug resistance gene . HOTAIR may contribute to the regulation and functioning of the PRC2 subunits. In addition to HOTAIR, a correlation between alterations in EED1 was also seen across the different cancer types [Figure HOTAIR on PRC2, chromatin remodeling and individual components, e.g., EED1, of PRC2.s Figure . Future H19 promoter [Theragnostics, a portmanteau of therapeutics and diagnostics, describes a system to customize healthcare using molecular and genetic tools for treatment decisions tailored to the individual patient. LncRNAs are therefore perfectly tailored as cancer theragnostics to design personalized medicines based upon molecular features of each tumor , 110. Nopromoter , 119. InHOTAIR, are deregulated in many cancer types, affect signaling pathways that promote cancer and modulate gene expression. Future studies will identify similar lncRNAs leading to signatures to better diagnose cancers. LncRNAs should allow for better stratification of cancer subtypes to correlate lncRNA expression with therapeutic response, drug resistance and overall survival. Similar to miRNA expression profiling in multiple myeloma and related B cell malignancies, lncRNAs profiling should lead to the identification of genetic subtypes and their association with treatment response and survival [LncRNAs are rapidly being recognized as important regulators of gene expression in cancer. The evolutionary conservation, diversity and complexity of lncRNAs indicates that they exert significant regulatory control on cell growth. While understanding the mechanistic role of lncRNA in cancer is still early, general principles are emerging. LncRNAs, e.g., survival –122. Fin


Internalization of gap junction plaques results in the formation of annular gap junction vesicles. The factors that regulate the coordinated internalization of the gap junction plaques to form annular gap junction vesicles, and the subsequent events involved in annular gap junction processing have only relatively recently been investigated in detail. However it is becoming clear that while annular gap junction vesicles have been demonstrated to be degraded by autophagosomal and endo-lysosomal pathways, they undergo a number of additional processing events. Here, we characterize the morphology of the annular gap junction vesicle and review the current knowledge of the processes involved in their formation, fission, fusion, and degradation. In addition, we address the possibility for connexin protein recycling back to the plasma membrane to contribute to gap junction formation and intercellular communication. Information on gap junction plaque removal from the plasma membrane and the subsequent processing of annular gap junction vesicles is critical to our understanding of cell-cell communication as it relates to events regulating development, cell homeostasis, unstable proliferation of cancer cells, wound healing, changes in the ischemic heart, and many other physiological and pathological cellular phenomena. Gap junctions are membrane channels composed of proteins termed connexins , 2. ThesIn this article, we will first discuss the morphological characteristics of annular gap junction vesicles as revealed over time with combined multi-morphological imaging techniques. Then, we will review the current knowledge on annular gap junction formation and subsequent processing including fission, degradation and potential connexin protein recycling. We also discuss the known cellular proteins that regulate these processing events.While the first transmission electron microscopic images of gap junction plaques were acquired in the early 1960s –31, it wThe first freeze fracture electron microscopic report describing annular gap junction vesicles was published in 1973 . With frIt should be noted however, that in early years, the existence of annular gap junction vesicles was met with controversy. Some investigators suggested that the profiles seen in TEM were only cross sections through invaginations from the cell surface , 51. HowFurther confirmation for the existence of annular gap junction vesicles rather than cross-sections of gap junction membrane invaginations came from lanthanum infiltration studies, which were used to demonstrate that the 2-4 nm “gap” of the annular gap junction membrane did not fill with lanthanum . The lacAnnular gap junctions were found in a number of different cell types  , 52–55 aWith the availability of antibodies directed against the various connexin family members, it was possible to use immunofluorescence microscopy to demonstrate the vast tissue distribution of annular gap junction vesicles. Immunofluorescence, compared to TEM or freeze fracture, is a relatively straightforward and inexpensive method for detecting gap junction plaques and annular gap junction vesicles. By using an anti-connexin antibody and a secondary antibody (which has been conjugated to a fluorophore) it was possible to visualize annular gap junctions with a fluorescence microscope , 58–61  but are not in contact . Three-dAnnular gap junctions and their associated proteins, detected with immunofluorescence have been accurately identified and correlated with quantum dot immuno-electron microscopy techniques. With quantum dot immuno-electron microscopy techniques, the characteristic gap junction membrane ultrastructure can be easily discerned and used to positively identify annular gap junction vesicles and their associated proteins Fig. . As seenThe field advanced further by the isolation of the jellyfish green fluorescent protein (GFP) , the couThe internalization of the gap junction plaque membrane is a critical step in the removal of gap junction plaques. Based on time lapse image analyses the gap junction plaque internalization process first involves the formation of a shallow curvature of the gap junction plaque , 61. Thi230VFF233, Y265AYF268, Y286KLV289) were identified in the connexin C-terminus (Cx43-CT), two of which (S2 and S3) were found to function cooperatively as AP-2 binding sites  connexon docking and channel accrual, and [3] transition of functional into permanently closed gap junction channels that then are [4] ‘primed’ to interact with clathrin to [5] mediate their endocytosis. These 5 steps also involve and require Cx43-ubiquitination and ZO-1 binding and release. All steps, including the critical posttranslational modifications are shown schematically in Fig. Note that the 5 transition steps described above group into two phases, “Early” (green) and “Late” (red) that are spatially separated on the Cx43 C-terminus Fig. . Early eChen and colleagues Cx43-peptide/PDZ-domain binding assays also revealed that the Cx43/PDZ-2 interaction requires that two ZO-1 proteins undergo a structural dimerization known as “domain swap” as they bind two separate Cx43 C-terminal peptides. Such dimerization results in a structural assembly that is believed to make the PDZ-2/Cx43 interaction Cx43 specific . NotablyAs gap junction plaque assembly and internalization consists of several steps, it furthermore is not surprising that multiple players and post-translational modifications are involved to achieve these events. In addition, implementing a succession of different types of modifications likely increases specificity and flexibility, including aborting and reverting the chain of events in case such changed cellular conditions call for increased GJIC. Continued research will be essential to further substantiate this elegant and elaborate regulatory machinery described by our model. [Note added in proof: During the typeset stage of this manuscript a review article suggesting a similar model of the internalization of gap junctions was published lending further support to our gap junction internalization model presented here .]In time-lapse recordings of living cells, internalized annular gap junction vesicles have been observed as translocating away from the plasma membrane and deeper into the cytoplasm , 61. StaMyosin-VI is the only motor protein known to migrate toward the pointed (minus) ends (located peri-nuclearly) of actin filaments . Myosin-Following internalization, annular gap junction vesicles have been reported to fragment or divide into smaller portions, or to bud smaller annular gap junction vesicles from a larger annular gap junction , 61 Fig. This frDynamin has been well documented to serve in the scissoring of numerous other types of non-junctional vesicles from membranes . SpecifiThe evidence that dynamin facilitates scissoring of the gap junction plaque bud from the cell surface and thus annular gap junction formation is based on colocalization immunofluorescence studies in which dynamin was demonstrated to associate with Cx43-GFP gap junction plaques , 61, andBy monitoring annular gap junction vesicles in control and dynasore treated cell populations and visualizing fission events by tracking the movement, morphological changes, and the size of individual annular gap junction vesicles over time, evidence has been provided that similar to its scissoring function in the release of gap junction plaque membrane buds, dynamin facilitates annular gap junction vesicle fission  Fig. 12. With tiFission of the annular gap junction vesicle poses a topological challenge in that it has a double membrane, and unlike the thin neck of the gap junction bud, it has a larger area that has to be constricted to achieve vesicle fission. Similar challenges are faced by mitochondria, which can be as large as or larger than annular gap junction vesicles. Mitochondria, like annular gap junction vesicles, are double membrane-bound organelles. During the mitochondrial fission process, two members of the dynamin superfamily, dynamin-related proteins (Dnm1) in yeast, and dynamin-related protein (DRP1) in mammals, have been demonstrated to assemble into punctate structures on mitochondria surfaces –170. TheCells have developed three principal degradation pathways: the proteasomal, the endo-lysosomal, and the phago-/lysosomal system (termed macroautophagy or simply autophagy) and all three serve specific cellular functions. The two latter degradation systems utilize the lysosome for final degradation. All three degradation systems have been implicated previously at various steps in the regulation of gap junction stability and connexin degradation , 173–178Several recent studies report the degradation of annular gap junctions by autophagy in situ as well as in cells in culture –20, 158 In addition, a large number of proteins essential or relevant for autophagosome formation and autophagic degradation (termed Atg-proteins) have been characterized and can be used as reliable markers for autophagic degradation. Of these, the ubiquitin-like proteins Atg12 (that is conjugated to Atg5) and LC3/Atg8, Atg7, and the PI3-kinase complex-component Beclin-1 (BECN1)/Atg6  are essential for autophagosome formation and autophagic degradation. All of them have been targeted and found important for annular gap junction degradation , 18–20, The ubiquitin-binding protein, p62 or sequestosome 1 (p62/SQSTM1) was found to colocalize with gap junction plaques in HeLa, COS7, and primary pulmonary artery endothelial cells , 18, 20.The endo-lysosomal system specifically degrades protein cargo that has been taken up at the plasma membrane in vesicles that mature into, or fuse with endosomes; or cargo that in other ways has entered endosomes. Membrane vesicles containing intact endocytosed gap junctions, or gap junctions apparently in the process of degradation have been observed in several ultrastructural studies , 192–194Implicit in the degradation of annular gap junctions through the endo-lysosomal pathway is the fusion of a double-membrane vesicle containing densely packed gap junction channels (an annular gap junction vesicle) with a single-membrane endosome. The mechanism by which this is achieved has not been elucidated. In several reports it has been suggested that the inner membrane of the annular gap junction splits away from the outer membrane, generating a single-membraned cytoplasmic annular hemi-gap junction vesicle (with the inner annular gap junction membrane remaining inside) and thus could fuse with the single-membrane endosome , 195–197Interestingly, TPA is a known potent activator of protein kinase C (PKC), and to promote hyper-phosphorylation and hyper-ubiquitination of Cx43 , 23, 110A recycling of annular gap junctions back to the plasma membrane has been recently suggested , 91, 158Carette and colleagues have, based on time-lapse imaging and transmission electron microscopic data, suggested that an entire annular gap junction vesicle may return to the plasma membrane to reform a gap junction plaque . In addiIn addition to the proposed recycling of annular gap junction vesicle connexins or their buds, VanSlyke and colleagues have reported that cytosolic stress, induced by heat shock (42 °C), which did not alter the endocytosis of biotinylated Cx43 but rather inhibited Cx43 degradation, increased intracellular levels of connexin proteins and increased the accumulation of connexins into gap junction plaques. Based on these results, they suggested that high levels of connexin within intracellular compartments following cytosolic stress may allow for increased opportunities for connexins to recycle back to the plasma membrane to participate in gap junction plaque formation . FinallyIt is now well accepted that gap junction internalization, which results in annular gap junction vesicle formation, is a major cellular pathway that significantly contributes to gap junction turnover and that this process utilizes the clathrin-mediated endocytosis machinery. Mutations in gap junction connexins can cause a number of devastating human diseases including inherited nonsyndromic hearing loss, X-linked Charcot-Marie-Tooth neuropathy, congenital eye lens cataracts, cardiac diseases such as hypertrophy, ischemia, and heart failure, a number of acute skin disorders, as well as craniofacial bone and other developmental defects (recently reviewed in ref. –202). ItExploring annular gap junction degradation processes via autophagosomal versus endo-lysosomal mechanisms appears important as well, as degrading annular gap junctions by different cellular mechanisms bears the potential for different outcomes; an interesting hypothesis especially when considering a potential re-use of connexin polypeptides, gap junction hemi-channels, or even entire gap junction channels, rather than their degradation. Finally, as gap junction biosynthesis is a complex, time-consuming and energetically costly process, the recently suggested concept that internalized gap junctions could be re-used by re-inserting them into the plasma membrane  is a pro


To investigate brain tumours and metastases many different radiopharmaceuticals imaged by SPECT and PET can be used. In this review the main and most promising radiopharmaceuticals available to detect brain metastases are reported. Furthermore the diagnostic contribution of the combination of SPECT and PET data with radiological findings  is discussed.Nuclear medicine techniques  represent molecular imaging tools, able to provide  Positron-emitting radiopharmaceuticals visualized by PET scan will be presented for first, as they represent the most valid and promising biomarkers to investigate brain tumours, while gamma-emitting radiocompounds, visualized by SPECT scan, will be discussed for the particular role played in the past, before the wide diffusion of PET tomographs. Finally findings obtained by multi-modality imaging combining nuclear medicine and radiological images will be reported.in vivo label biochemical processes and metabolic pathways , comparing with gamma-emitting radiopharmaceuticals able to indirectly enter in a specific metabolic function . However positron-emitting radiocompounds have some disadvantages, because their production depends on the presence of an on-site cyclotron and the final process is expensive fluoro-d-glucose (18FDG), a using glycolytic pathways, is the most widely used because it is available in all the PET Centres. Other more tumour-selective but less diffuse radiopharmaceuticals will be also reported in this review.Among the group of positron-emitting radiolabelled compounds 2-deoxy-, while early or late SUVs of the lesion alone did not distinguish between tumour and necrosis. Furthermore differentiation between necrosis and metastatic brain disease was improved by using the change of lesion to gray matter SUVmax ratios as a function of time independently of histological tumour type. The limit is the long time interval between PET scans  as suggested also by other authors  was capable to separate the two groups (p < 0.001). The authors concluded that a three-compartment model with blood volume, metabolite and partial volume corrections may adequately describe 18FLT kinetics in malignant brain tumours. Furthermore patients could be divided as having tumour-predominant or treatment change-predominant lesions, showing significantly different phosphorylation rates.A further interesting radiopharmaceutical is 3'-deoxy-3'- tissues . 18FLT hs et al.  investigin vivo biomarkers to image brain tumours and metastases, for the mentioned ability to in vivo label biochemical and metabolic pathways the gamma-emitting radiocompounds selective for neuro-oncology played a relevant role in the past and can be still used. Gamma-emitting radiopharmaceuticals are visualized by SPECT scan and are commercially available, thus not requiring the presence of the on-site cyclotron. However the resolution power of SPECT is lower than that of PET tomograph. Many radiopharmaceuticals have been used to study brain tumours: 201Thallium (201Tl), 99mTc-sestamibi (99mTc-MIBI), 99mTc-tetrofosmin and 123I-iodine-α-methyl tyrosine (123IMT) methyl-l-tryptophan (AMT)-positron emission tomography (PET) to differentiate newly diagnosed glioblastomas and brain metastases in 36 adults with suspected brain malignancy. AMT is an amino acid positron-emitting promising radiopharmaceutical tracer able to measure tryptophan metabolism via the immunomodulatory kynurenine pathway [et al. [A further interesting example of multi-modality imaging is represented by the paper of Kamson et al. , that ev pathway . Althoug pathway ,44. Kams [et al.  measuredet al. [i.e., SPECT and PET, diagnostic accuracy was improved [Finally Kickengereder et al.  reportedet al. . On the improved ,32, thusin vivo biomarkers of primary and metastatic brain tumours. PET with positron emitting tumour selective radiopharmaceuticals represents the option of choice to obtain the best diagnostic accuracy. 18FDG PET can be useful if brain lesions show clearly higher or lower tracer uptake comparing with normal tissues or if fused images with radiological tools providing anatomic location of tumour are available. The association of nuclear medicine and radiological modalities can contribute to improve diagnostic accuracy by combining morphological and functional information.In conclusion nuclear medicine techniques represent molecular imaging tools able to provide Finally it has to be remarked that in international literature many papers are available on the role of molecular imaging modalities in detecting primary brain tumours, while data concerning brain metastases are relatively scarce, although the published papers present very promising results. More research works are necessary to support these findings.


The most promising approach follows the strategy of early detection of tumor progress prior to clinical manifestation in prostate gland. The PSA (prostate specific antigen) is used as the universal marker in clinical practice since 1979 year, however, with modest success. Hence, 40% of prostate cancer patients demonstrate normal levels of PSA , and only 60% of the patients have higher levels of PSA . Consequently, the world research community further efforts to develop more specific and sensitive markers for early detection of pre-cancerous lesions in prostate gland. A promising tool might be “omics” and in particular “proteomics” technologies. Predictive, Preventive and Personalized medicine is considered as the medicine of XXI century with following requirements: risk assessment, disease specific profiles, early/predictive diagnostics, disease/treatment monitoring, reliable prognosis, and treatments tailored to the person.Cancer of prostate is challenging medical issue which prevalence ranking in men among all oncologic diseases corresponds to the 4th most prevalent in Russia, 2development of a genomic-proteomic system for early diagnosis and clinical monitoring of patients with prostate cancer as follows:• To determine a genome-wide significant association between PSA level and single-nucleotide polymorphisms (SNPs) at loci;• To define potential candidates for blood peptide's biomarker expression database, as the platform for the identification, quantification and validation of protein biomarker(s) during prostate cancer progression;• To create a centralized prostate cancer urine peptide's biomarker expression database with add value for the blood biomarkers database;• To analyze diagnostic accuracy of the prostate cancer biomarker candidates;• To create innovative approaches for risk assessment, therapy monitoring and treatment management for this patient cohort.With altogether over 1000 prostate cancer patients a robust database has been created in the study for follow-up molecular biological analysis. To estimate a genome-wide significant association between PSA level and single-nucleotide polymorphisms (SNPs) at loci, MALDI-TOF based mini-sequencing of blood samples is utilized.The sequencing data will be correlated with other parameters such as individual PSA levels, cancer stage and tumor aggressiveness. In the next steps, blood and urine profiles will be analyzed at the level of proteins. Multi-factorial analyses are expected to reveal correlated between individual parameters. Creation of the genome-transcriptome-proteome patterns is the promising approach to establish early detection, predictive diagnostics, reliable prognostics and improved management of prostate cancer.


Typically, joint arthroplasty is performed to relieve pain and improve functionality in a diseased or damaged joint. Total knee arthroplasty (TKA) involves replacing the entire knee joint, both femoral and tibial surfaces, with anatomically shaped artificial components in the hope of regaining normal joint function and permitting a full range of knee flexion. In spite of the design of the prosthesis itself, the degree of flexion attainable following TKA depends on a variety of factors, such as the joint's preoperative condition/flexion, muscle strength, and surgical technique. High-flexion knee prostheses have been developed to accommodate movements that require greater flexion than typically achievable with conventional TKA; such high flexion is especially prevalent in Asian cultures. Recently, computational techniques have been widely used for evaluating the functionality of knee prostheses and for improving biomechanical performance. To offer a better understanding of the development and evaluation techniques currently available, this paper aims to review some of the latest trends in the simulation of high-flexion knee prostheses. Many daily activities require considerable knee flexion, level walking >60°, ascending stairs >80°, sitting >90°, and getting out of a bath >130° . High-flThe choice to use a high-flexion knee is ultimately left to the surgeon, with patient consent. As such, this report will compile published data regarding the biomechanical aspects of HF total knee replacement (TKR) with special focus on posterior cruciate ligament retaining knees (cruciate-retaining (CR)) and posterior cruciate ligament sacrificing designs (posterior-stabilized (PS)). Meta-analyses comparing CR and PS knees have generally shown no favourable design in terms of longevity, range of motion, and pain. A recent Cochrane Database review  of 17 stThis review details two computational methods for studying the biomechanics of knee prostheses. One method uses multibody dynamics software, namely, MSC Adams , to study the dynamic behaviour of the knee joint. Another technique is to use finite element analysis (FEA) to study the internal mechanical condition of the joint: stress, strain, and so forth. The FEA research detailed below typically used ABAQUS  accompanied by some preprocessing and meshing software.The major drawback in the computational simulation of joints lies in the simplifications that must be made within each model. Some of these assumptions are purposely designed into the model in consideration of cost, computing power, time, processing, and so forth, but it must also be noted that such simulations have not advanced far enough to realistically imitate all of the components and tissues within the joint. Inherent simplifications in the modelling process, such as inaccurate representations of soft tissues, need to be taken into consideration. The validity of a model may be determined by comparing the results against in/ex vivo studies of a representative human joint or by comparing the models against previously validated simulations. Each of the models detailed below has been validated in this manner.High knee flexion >120°) requires significant translation of the femoral condyles on the tibial plateau. Inadequate femoral rollback and tibial rotation are common complications with current high-flexion prostheses during such high flexion 0° requir. To overRetrieval studies have consistently shown incongruent articular surfaces to be associated with a greater risk of polyethylene wear –21. IncrAdditionally, mobile bearing tibial inserts have been developed to offer dual articulation in the knee, theoretically permitting a greater range of motion and reducing contact stress, and in turn reducing wear , 23. In It has been reported that at least 19% of patients receiving posterior stabilized (PS) knees suffer abnormal tibiofemoral axial rotation . While tIn a related study, Huang et al. analysed the stress on the tibial post for flat-on-flat and curve-on-curve designs up to a knee flexion angle of 150° . Two conIn a bid to further improve tibial rotation, Lin et al.  modifiedIn conclusion, these studies promote the use of anatomically shaped knee prostheses to achieve high knee flexion while limiting component wear. A rounded and convex lateral plateau and a shallower medial plateau on the tibial surface promote femoral rollback and permit more natural knee motion. Also, the post-cam contact surfaces should be rounded on both the anterior and posterior faces to reduce edge loading and produce a greater contact surface area throughout flexion. As mentioned, an asymmetric curve-on-curve cam shape may improve tibial rotation but at the expense of femoral rollback.


Plastids are actively involved in numerous plant processes critical to growth, development and adaptation. They play a primary role in photosynthesis, pigment and monoterpene synthesis, gravity sensing, starch and fatty acid synthesis, as well as oil, and protein storage. We applied two complementary methods to analyze the recently published apple genome  to identify putative plastid-targeted proteins, the first using TargetP and the second using a custom workflow utilizing a set of predictive programs. Apple shares roughly 40% of its 10,492 putative plastid-targeted proteins with that of the Arabidopsis  plastid-targeted proteome as identified by the Chloroplast 2010 project and ∼57% of its entire proteome with Arabidopsis. This suggests that the plastid-targeted proteomes between apple and Arabidopsis are different, and interestingly alludes to the presence of differential targeting of homologs between the two species. Co-expression analysis of 2,224 genes encoding putative plastid-targeted apple proteins suggests that they play a role in plant developmental and intermediary metabolism. Further, an inter-specific comparison of Arabidopsis, Prunus persica (Peach), Malus × domestica (Apple), Populus trichocarpa (Black cottonwood), Fragaria vesca (Woodland Strawberry), Solanum lycopersicum (Tomato) and Vitis vinifera (Grapevine) also identified a large number of novel species-specific plastid-targeted proteins. This analysis also revealed the presence of alternatively targeted homologs across species. Two separate analyses revealed that a small subset of proteins, one representing 289 protein clusters and the other 737 unique protein sequences, are conserved between seven plastid-targeted angiosperm proteomes. Majority of the novel proteins were annotated to play roles in stress response, transport, catabolic processes, and cellular component organization. Our results suggest that the current state of knowledge regarding plastid biology, preferentially based on model systems is deficient. New plant genomes are expected to enable the identification of potentially new plastid-targeted proteins that will aid in studying novel roles of plastids. Arabidopsis thaliana) and cyanobacteria indicates that 18% of the Arabidopsis protein–coding genes were derived from events involving transfer of genetic material from the plastid to the nucleus The plastid is an intracellular organelle derived from an endosymbiotic event wherein a free-living autotrophic photosynthetic bacterium was phagocytized by a separate heterotrophic organism Plant cells have developed intricate mechanisms to import nuclear-encoded proteins to or across the three plastid membranes . The presence of multiple protein transport pathways have been shown to play a role in aiding protein transport across the inner and outer plastid envelopes; however, the vast majority of the plastid proteome is transported via the tic/toc pathway Due largely to the technical complexity with whole plastid proteome characterization, transcriptome or genome sequences have become a widely used dataset to predict plastid-targeted motifs. Such an approach also enables the identification of plastid-targeting proteins in a spatial and temporal context. Prediction of subcellular localization has been reported to be performed with software such as PCLR The Rosaceae family represents a unique diversity in fruit development, which is unrealized in the many model plants whose genomes have been sequenced The plastid structure has also been reported to differ between different tissues of the fruit. Phan In this study we tested the three primary hypotheses using a bioinformatics approach, (1) The total number and composition of the plastid-targeted protein coding genes in apple, a model representative of Rosaceae, that is taxonomically different from Arabidopsis, (2) The plastid-targeted protein coding genes are under transcriptional control during apple fruit development and (3) There is a subset of unique plastid-targeted protein coding genes that are unique and novel to each plant species.Arabidopsis thaliana, Vitis vinifera, Prunus persica, Populus trichocarpa, Fragaria vesca, and Solanum lycopersicum resulting in the identification of plastid-targeted proteins unique to each species. A core set of 737 Arabidopsis thaliana proteins, highly enriched in photosynthesis and primary metabolism gene ontology (GO) terms, were identified to have homologous plastid-targeted proteins in all investigated species.In order to test the first hypothesis, we performed an in-depth computational analysis predicting the plastid-targeted proteome of apple and compared it with Arabidopsis resulting in the identification of a much larger number of plastid-targeted genes with nearly 4000 plastid-targeted protein coding genes being unique to apple. The second hypothesis was tested by reanalyzing publically available apple transcriptome data which revealed the presence of co-expression profiles of plastid-targeted genes and their association to development and metabolism. Finally, the third hypothesis was tested by extending the custom analysis workflow to an inter-genera comparison between six published genomes: Malus × domestica predicted protein set was obtained from the apple genome sequencing project The A part of the functional annotation pipeline was applied to identify organelle ‘plastid’ targeted gene products encoded by the apple genome Fragaria vesca genome paper Arabidopsis thaliana, Brachypodium distachyon, Caenorhabitis elegans, Chlamydomonas reinhardtii, Danio rerio, Eschericia coli, Fragaria vesca, Glycine max, Homo sapiens sapiens, Zea mays, Malus × domestica, Mus musculus, Neurospora crassa, Oryza sativa, Physcomitrella patens, Populus trichocarpa, Saccharomyces cerevisiae and pombe, Selaginella moellendorffii, Sorghum bicolor, Synechosystis, and Vitis vinifera to cover the tree of life with emphasis on fully/nearly complete and published genomes. The peptide sequences were downloaded from Phytozome.net for grapevine, Selaginella, Physcomitrella, Chlamydomonas, Glycine, Populus, and Malus from the genome portal GrameneThe Inparanoid algorithm Arabidopsis thaliana plastid-targeted gene set was obtained from the Chloroplast 2010 project website (www.plastid.msu.edu) Arabidopsis thaliana using USEARCH M. × domestica dataset were compared between the TargetP-based method as well as the custom analysis to investigate any bias associated with either respective prediction technique.The Fragaria vesca (Woodland Strawberry) Vitis vinifera (Grapevine) Solanum lycopersicum ITAG1 release (Tomato) Prunus persica (Peach) Populus trichocarpa (Black Cottonwood) Arabidopsis thaliana plastid-targeted dataset, the entire Arabidopsis TAIR V10 protein set (Arabidopsis.org) Solanum lycopersicum, as well as a file comprised of the sequences of the predicted plastid-targeted protein sequences of the other six species. All datasets were first sorted by length using USEARCH.Predicted coding sequences were collected from the genomes of Arabidopsis thaliana putative plastid-targeted protein set was compared using USEARCH 40/40 global parameters separately against the plastid-targeted proteins from woodland strawberry, grapevine, tomato, peach, black cottonwood, and apple. Output files were then analyzed to identify those Arabidopsis sequences which had a match in the plastid-targeted proteomes of all species.Further analysis was performed with USEARCH to identify those proteins present in the predicted plastid proteomes of all investigated species. To perform this analysis, the A second comparative analysis was performed using the clustering feature of the USEARCH package, UCLUST. In this analysis, the plastid-targeted protein sequences from the seven examined species were compiled into a single file and sorted by length. UCLUST was performed at 50% identity. The output was parsed to identify protein clusters with members from all seven species, as well as those clusters containing sequences from only one species.Two separate techniques were used to create the similarity matrices based upon Jaccard's coefficients. To calculate the value of an individual cell (the distance between species A and species B) we first determined if two genes were considered homologous. If a gene from the species A matches with another gene in species B, then both A and B are included in the intersection set. The result in the cell is the count of the intersection set divided by the sum of all the genes in both species. For the USEARCH-based approach, two genes match if they align to each other using 40/40 parameters. Alternatively, in the UCLUST-based approach two genes match if they belong to the same cluster.Sequences for all genes encoding unique or shared plastid-targeted proteins in the investigated apple, Arabidopsis, grapevine, peach, strawberry, black cottonwood, and tomato datasets were analyzed via Blast2GO In order to ascertain if genes encoding plastid-targeted proteins in apple are expressed in fruits, as well as to identify co-expressed gene sets, microarray data from a previously published experiment were used Arabidopsis thaliana , plastid-targeting predictions were primarily performed using Target P. TargetP was selected in order to be consistent with previously published work in this area and because previous studies have found TargetP to be the most reliable single prediction program Malus × domesticawith 18.3% of its nuclear-encoded proteins predicted to be plastid-targeted, while the lowest was that of Populus trichocarpa with only 9.9%  Arabidopsis plastid-targeted proteome subset, while about 60–70% of the proteins have likely homologs in the entire Arabidopsis protein subset  than they had in the Arabidopsis proteome (40–60%)  . StrawbePrunus persica to 41.5% in Fragaria vesca in the USEARCH method and 20.6% in Prunus persica to 46.8% in Fragaria vesca in the UCLUST method. However, the majority of the protein sequences have a homolog in at least one of the investigated species  was enriched in apple, Arabidopsis and tomato. Cell death (GO:0008219) and death (GO:0016265) were also enriched in the apple UCLUST50 dataset.GO term enrichment analysis was performed on the species-specific plastid-targeted protein sequences for both UCLUST and USEARCH-based analyses. Comparisons were performed using a Fisher's test with the entire predicted plastid proteome as a reference. This analysis revealed significantly enriched GO terms for both comparative techniques. In the USEARCH based technique, the majority of enriched GO terms were present in the unique apple plastid-targeted sequences with the most significant GO terms were DNA metabolic process  and cellular macromolecule metabolic process (GO:0044260) . AdditioArabidopsis appear to be the best characterized with 98% containing some form of GO information, followed by those of Prunus persica with 81.9%. Solanum lycopersicum displays the least amount of GO information with only 56.5% of these uniquely plastid-targeted proteins having associated GO terms.While a significant amount of functional annotation was performed with Blast2GO, a large proportion of the proteins predicted to be unique to each species lack any associated GO term . ProteinArabidopsis thaliana was compared with every other species’ predicted plastid proteome at 40% identity and 40% coverage. Those proteins which have a matched protein from all other six species were then determined to be conserved across the plastid proteomes.Two separate analyses were performed to identify proteins which were predicted to be targeted to the plastids of all seven angiosperms studied. First analysis workflow utilized a semi-global approach where UCLUST Arabidopsis thaliana, 773 from Malus × domestica, 384 from Vitis vinifera, 392 from Fragaria vesca, 545 from Populus trichocarpa, 439 from Prunus persica, and 478 from Solanum lycopersicum. Blast2GO analysis reveals that these proteins are involved in a large number of biological processes , response to stress , and carbohydrate metabolic process   . Again, roteins) . GO termroteins) . The lowArabidopsis thaliana plastid-targeted proteomes. However, USEARCH40/40 enriched GO terms contain an additional 10 biological process not represented in the UCLUST50 analysis; anatomical structure development (GO:0009653), regulation of biological quality (GO:0065008), anatomical structure morphogenesis (GO:0009653), reproductive process (GO:0022414), cell growth (GO:0016049), regulation of anatomical structure size (GO:0090066), regulation of cell size (GO:0008361), regulation of cellular component size (GO:0032535), homeostatic process (GO:0042592), and cellular homeostasis (GO:0019725).Comparing the USEARCH40/40 dataset along with that of the UCLUST50 dataset reveals that the two methods agree upon 439 shared plastid-targeted sequences, with 58 present only in UCLUST50 and 298 in USEARCH40/40. Both datasets have similar enriched GO terms with 49 enriched compared to the entire Arabidopsis thaliana loci from the UCLUST50 dataset and the 737 protein sequences from the USEARCH40/40 dataset along with their respective GO terms are provided in The UCLUST50 enriched GO terms include five biological processes  and two cellular components [cytoplasmic part (GO:0044444) and cytoplasm (GO:0005737)] not represented in the USEARCH40/40 analysis. A complete list of the 478 Arabidopsis thaliana  were compared with those identified as shared between the seven species examined in this study both with USEARCH 40/40, as well as UCLUST 50% using Venny Proteins conserved in this study were further compared with those from GreenCut2. GreenCut2 represents a collection of 597 nuclear-encoded proteins determined to be conserved across 20 photosynthetic eukaryotes, but absent in non-photosynthetic organisms Arabidopsis. An additional modified Jaccard similarity coefficient matrix was generated with the UCLUST50 analysis. In this matrix the predicted plastid-targeted of all species are most similar to that of peach, while that of peach is most similar to apple.In order to look at the overlap of homologous proteins between each species, the Jaccard similarity coefficient was determined for the USEARCH40/40 . This coArabidopsis, 737 have significant identity to predicted plastid-targeted proteins in each of six investigated species, suggesting an evolutionarily core conserved set of plastid-targeted proteins. The caveat is that TargetP accuracy is not well defined, and has been shown to differ between experiments Arabidopsis thaliana chloroplast proteome resulting in the identification of 604 nuclear-encoded proteins Arabidopsis thaliana chloroplasts identified through MALDITOF MS and nano-LC-ESI-MS/MS had a much higher predictability to be chloroplast targeted by TargetP with 88% accuracy Arabidopsis plastid-targeted proteins revealed that 86% were correctly predicted using TargetP This study reveals several interesting aspects about the constitution of predicted plastid-targeted proteomes for the species analyzed. A large portion of a plant’s nuclear genome is dedicated to plastid-targeted proteins, many of which lack identity to plastid-targeted proteins in other species. Some plastid-targeted proteins have identity with potentially alternatively-targeted proteins in other systems. Of the predicted plastid-targeted proteins in Arabidopsis and Oryza sativaArabidopsis chloroplast proteome containing 2,100 proteins shared only 900 with the 4,800 plastid proteins in Oryza sativa. These 900 proteins were largely involved in transcription, energy, and metabolism. It would not be surprising to see this shared set of proteins shrink substantially as the number of species compared increases. A large focus has been put on the identification of proteins found only in photosynthetic organisms termed the GreenCut While it may be expected that plastid-targeted proteins would be highly conserved in this 7-species analysis, a previous study demonstrated a striking lack of similarity between the plastid proteomes of −10Chalydomonas and Arabidopsis and human to identify paralogs, co-orthologs, and orthologs of Chlamydomonas−10 to identify orthologs and included sequences with over 50% amino acid identity as in-paralogs Differences in the outcome of comparative analysis projects can be attributed to the utilization of alignment or clustering methods. Previous studies in microbial comparative proteomic and genomic studies have utilized an alignments of 50% identity and 50% coverage to predictively resolve paralogs from orthologs The unique plastid-targeted proteins within each of the investigated species possess varied levels of functional information. Despite the lack of spatial and temporal transcriptome and proteome expression context, this information has a large referential value for future work. Blast2GO analysis and GO term enrichment analysis provide a glimpse as to what these proteins are likely participating in within the plastids. However, only a few GO terms were found to be enriched, most of which differed between species. This suggests that there are likely not specific classes of proteins which fall into this category of “unique” for the plastid proteomes of each species. As expected, due to substantial research performed on it, a large proportion of the proteins unique to the Arabidopsis plastid proteome (98%) possess associated GO terms . HoweverPlastids play an integral part in plant development, photosynthesis, and several other known biochemical processes. However in fruits their role has remained uncharacterized. Several important biochemical processes for synthesis and storage of pigments, nutraceutical and medically important compounds as well as aromatic compounds are resident in fruit plastids. These components are important both for consumer appeal as well as nutritional value. In addition, various physiological disorders, such as sunscald in apple, are associated with the inability of fruits to adequately quench excessive energy from sunlight. Through furthering our understanding of the plastid function in non-model plant systems and organs such as fruit, novel mechanisms for enhancing photosynthetic efficiency and crop productivity could be discovered.The results presented in this work indicate that the current state of knowledge regarding plastid biology, mostly derived from model systems, is not comprehensive enough. In each plant species evaluated in this work, plastids are predicted to host a plethora of biological and metabolic processes necessitating subsequent wet-lab validation in non-model systems. New plant genomes are expected to enable the identification of potentially new plastid-targeted proteins that will aid in studying novel roles of plastids in plant development, metabolism and adaptation.While previous studies have advocated the integration of multiple protein localization prediction techniques The TargetP-based analysis indicates that a large subset of a plant’s nuclear-encoded proteome is predicted to be localized to the plastid. However, the proportion of transcripts encoding plastid-targeted proteins varies, compromising 10–20% of the transcriptome depending upon the species investigated. Of the nuclear-encoded plastid proteome, there appears to be a significant subset that is species-specific. Many of these proteins have homology to proteins not predicted to be plastid-targeted in other systems, indicating that it may be common for proteins to gain or lose targeting peptides during evolution. If this is indeed the case, it would be interesting to investigate the evolutionary and mechanistic context of gain or loss of target peptides across species.Through using two comparative methods, a USEARCH-based approach as well as a semi-global UCLUST-based approach, we displayed that very few plastid-targeted proteins are conserved between the predicted nuclear-encoded plastid proteomes. This value varied based upon the comparative technique and user parameters, but our predictions identify 497 and 737 Arabidopsis proteins which contain predicted plastid-targeted homologs in all other examined angiosperms. GO term enrichment analysis suggests that specific functions are significantly conserved in plastids, namely photosynthesis, many metabolic processes, transport, and cell death. Knowledge about these conserved proteins can be utilized in future studies to better understand and potentially predict those proteins which are plastid-targeted in other non-model systems and additionally identify novel plastid-targeted proteins.The expression of genes encoding plastid-proteins appears to be very diverse within the fruit developmental continuum with 64 significant expression patterns detected (those containing five or more genes). However, most of the genes investigated can be clustered into nine expression patterns. These expression patterns can be overlapped with important milestones within the development of fruit to find plastid proteins which may be responsible for novel fruiting milestones or processes. While expression data is available for ∼13,000 genes, subsequent developmental transcriptomics, metabolomics, and proteomics investigations are expected to provide a comprehensive understanding of the roles of plastids in apple fruit development.File S1Expression data from Janssen et al. for Malus × domestica genes predicted to encode plastid-targeted proteins. Expression data for each cluster of co-expressed genes with sequence header and expression data in the form of Log2 of expression relative to the lowest value.(XLSX)Click here for additional data file.File S2GO term information for Malus × domestica plastid-targeted protein clusters.(XLSX)Click here for additional data file.File S3Header information for predicted plastid-targeted proteins from the investigated seven species. Excel file containing identifiers of predicted plastid-targeted protein sequences generated through with TargetP analysis.(XLSX)Click here for additional data file.File S4Plastid-targeted proteins unique to each examined species with GO term information. Excel file containing the header information and associated GO terms.(XLS)Click here for additional data file.File S5Plastid-targeted proteins shared between all species. Excel file containing the header information for the UCLUST50 analysis separated by species. Information is provided for the 737 Arabidopsis thaliana sequences shared in the USEARCH comparative analysis.(ZIP)Click here for additional data file.File S6Blast2GO annotation file containing header and GO term information for 70 protein sequences present in GreenCut2, UCLUST50, and USEARCH4040 analyses.(ZIP)Click here for additional data file.


Proteobacteria  were the dominant taxa in the microbialites, and possessed novel functional guilds associated with the metabolism of heavy metals, antibiotic resistance, primary alcohol biosynthesis and urea metabolism; the latter may help drive biomineralization. Urea metabolism within Pavilion Lake microbialites is a feature not previously associated in other microbialites. The microbialite communities were also significantly enriched for cyanobacteria and acidobacteria, which likely play an important role in biomineralization. Additional findings suggest that Pavilion Lake microbialites are under viral selection as genes associated with viral infection  are abundant within the microbialite metagenomes. The morphology of Pavilion Lake microbialites changes dramatically with depth; yet, metagenomic data did not vary significantly by morphology or depth, indicating that microbialite morphology is altered by other factors, perhaps transcriptional differences or abiotic conditions. This work provides a comprehensive metagenomic perspective of the interactions and differences between microbialites and their surrounding environment, and reveals the distinct nature of these complex communities.Modern microbialites are complex microbial communities that interface with abiotic factors to form carbonate-rich organosedimentary structures whose ancestors provide the earliest evidence of life. Past studies primarily on marine microbialites have inventoried diverse taxa and metabolic pathways, but it is unclear which of these are members of the microbialite community and which are introduced from adjacent environments. Here we control for these factors by sampling the surrounding water and nearby sediment, in addition to the microbialites and use a metagenomics approach to interrogate the microbial community. Our findings suggest that the Pavilion Lake microbialite community profile, metabolic potential and pathway distributions are distinct from those in the neighboring sediments and water. Based on RefSeq classification, members of the  Microbialites are a specialized group of microbial mats that lithify carbonate-rich structures, and include thrombolites that consist of structures with unlaminated clots and stromatolites that have laminated layers . Fossil Microbialites are globally distributed, and can be found in marine , freshwaA host of biological factors are favorable to microbialite formation, such as the presence of exopolysaccaride (EPS)-rich cyanobacterial mats, which serve as a location of mineral nucleation and provide a heterotrophic microenvironment favorable for organomineralization via dissimilatory sulfate reduction . CyanobaDespite the apparent reliance of microbialites on biotic input from the surrounding environment, there is currently a scarcity of data comparing microbialite communities with those of the sediments or water. Such a comparison allows for the identification of microbialite-specific components that may not be obvious when examining microbialite communities in isolation. Exploring the genetic differences between microbialite communities and those in the surrounding habitats requires identifying the relative abundance of taxa and their metabolic potential. To avoid distorting these ratios, DNA was extracted and unlike in other metagenomic studies of microbialites , sequencMany studies have focused on examining the abundance and diversity of freshwater microbialites using 16S rDNA sequencing but few metagenomic studies exist. Diversity studies using 16S rDNA amplicons on freshwater microbialites include Lake Van , Lake Al-1), and oligotrophic . Further limnological details of Pavilion Lake are given in Figure 1; Sampling was conducted in Pavilion Lake, in southeastern British Columbia, Canada . The lake is dimictic, circumneutral  during the summers of 2010 and 2011. Triplicate representative microbialites (∼10 kg), were recovered from each collection site  either bin situ temperatures until samples were processed. At the field lab, each microbialite was weighed, documented and apportioned for molecular analysis. Replicate sediment samples were pelleted by centrifugation (5000 × g). The overlying lakewater was removed and the sediment pellets flash frozen in liquid nitrogen and transported back to the lab in a liquid nitrogen vapor shipper for downstream processing.Sediment samples, adjacent to the microbialites  were collected into sterile bottles at the same time by divers. At the lake surface, microbialite and sediment samples were immediately placed into insulated containers filled with cold lakewater to maintain ® screening to remove large plankton, and 1.2-μm pore-size glass-fiber, followed by 0.45 and 0.22-μm pore-size Durapore polyvinylidene difluoride (PVDF) filters   was collected from each depth using a Niskin water sampler (2010) or a diver guided hose at the collection site that was connected to a piston-pump in a boat (2011). Surface water samples (∼1 m depth) were collected using a submersible pump. Each water sample was filtered in series through 120-μm pore-size NitexMA, USA) . Filters® Soil DNA Isolation Kit  then flash frozen in liquid nitrogen. Replicate microbialite scrapings were placed into sterile jars and frozen on-site in liquid nitrogen. Frozen samples were transported back to the lab in a liquid nitrogen vapor shipper and stored at –80°C until needed. DNA from frozen samples were extracted using cetyl trimethyl ammonium bromide  across the surface of three morphologically similar microbialites collected at each depth. DNA was extracted on-site using a PowerMaxde CTAB; .® DNA Isolation Kit . DNA was extracted from the other half of each filter using the CTAB method , DNA was extracted from half of each glass-fiber, 0.45 and 0.22-μm pore-size filter using a PowerWaterB method .® Soil DNA Isolation Kit . Replicate sediment pellets were also extracted using the CTAB method. Two DNA extraction methods were employed for all samples to minimize extraction biases.Sediment DNA was extracted from triplicate subsamples (∼5 g) using a PowerMaxTM PicoGreen® dsDNA Assay Kit . Nucleic acid quality was determined by absorbance (260/280 and 260/230) using a Nanodrop-1000 . CTAB and MoBio DNA extractions were pooled (50:50) by equivalent DNA to reduce extraction bias and then used for library construction.DNA concentrations were determined on-site using a Nanodrop-3300 micro-fluorospectrometer and the Quant-iTLibraries for 454 FLX Titanium sequencing were constructed using random DNA shearing with a Bioruptor . Fragments were polished and blunt-end ligated  to in-house Multiplex Identifier barcode oligos , with small fragments removed by magnetic beads . The libraries were quantified using a digital PCR quantified standard curve , dilutedFor Illumina library construction, DNA was sheared by ultrasonication , and the fragments end-paired, A-tailed  and ligated to TruSeq adapters ; small fragments were removed twice using magnetic beads . The resulting libraries were pooled, and sequenced using both 250 bp and 100 bp paired-end sequencing on the MiSeq  and HiSeq  platforms, respectively.1  using a custom Perl script. Barcodes were removed by Tagcleaner  and sequTable 1), respectively. Surface (∼1 m) and 10 m water metagenomic reads were pooled at assembly step to yield >15 k contigs for further comparison. Sediment metagenomic data  resulted in a low numerical (<15 k) yield of contigs; hence, the unassembled paired-end reads were extended for overlap and pooled with unextended reads for further analysis (Table 1). Metagenomic rapid annotations using MG-RAST were used for contig annotation  using the Ray DeNovo assembler , 2012. Inotation . MG-RAST4 annotations were provided by MetaPathways, a modular pipeline for gene prediction and annotation that uses pathway tools and the MetaCyc database to construct environmental pathway/genome databases , an effect size (Eta-squared) and multiple test correction using Benjamini-Hochberg FDR  procedure. The RefSeq and SEED classifications were normalized for each sample using count-relative abundances and total ORFs obtained per metagenome. PCA for the normalized RefSeq and SEED classifications used R libraries Ecodist , and pvclust  using ward clustering and the Bray-Curtis distance matrix at a thousand replicates  and R . STAMP aplicates . The PCAplicates .5. The data is deposited in the project name Pavilion Lake surrounding environment as PLsfcFil (ID 4532785.3), PL20Fil (ID 4532783.3), PL45Fil (ID 4532784.3), PLMB10 (ID 4532771.3), PLMB20 (ID 4532772.3), PLMB25 (ID 4532774.3), PLMB45 (ID 4532775.3), PL10Sed (ID 4526738.3), PL20Sed (ID 4526739.3), and PL25Sed (ID 4526740.3).All the data used in this study is freely available from MG-RASTFigure 2A), SEED , and MetaCyc functional gene assignments. STAMP ANOVA of the RefSeq classifications identified thirteen bacterial classes that were significantly enriched in microbialites over the surrounding environment . ANOVA using STAMP on the highest level classification (level I) in the SEED database indicates that membrane transport, aromatic metabolism, motility, potassium metabolism, cell signaling and virulence genes are significantly enriched in microbialites over the surrounding environment . MetaCyc functional gene assignments suggest many shared pathways (263) among samples from the water (filters), microbialites and sediments with 246 pathways distinct to Pavilion microbialites . These observations support the idea that microbialite associated microbes are distinct and are not being seeded or introduced (at least not recently), from surrounding environments.The microbial community structure and metabolic potential of Pavilion Lake microbialites were statistically different from those in the surrounding environment , based on principle component analyses of RefSeq taxonomic classifications . Although the depth of sequences was not the same across microbialites, sediments and water, it was adequate to clearly show that the microbialite community was distinct from those in the surrounding environments. These findings are consistent with previous works that demonstrate fundamental differences in microbial taxa between microbialite-associated communities and others. The microbial communities and metabolic potential of the microbialites differed between the sediment and water samples. Compared to the microbialites, the sediment metagenomic data had more sequences assigned to the taxonomic groups Figure 2A) and SEED  classifications indicate that the microbial community and metabolic potential of microbialite metagenomes cluster closely together, regardless of morphology or depth . Across morphologies, >80% of the MetaCyc pathways predicted by the microbialite metagenomes are shared , with few (<30) distinct pathways within a Pavilion Lake microbialite morphotype.Microbialite morphology in Pavilion Lake changes predictably with depth, however the metabolic potential and microbial community remains similar. PCA of RefSeq . For example, sequences assigned to the classes Alphaproteobacteria, Deltaproteobacteria, Acidobacteriia, and Gloeobacteria were significantly more abundant in microbialite metagenomes than in the water or sediment metagenomes . The dominance of sequences associated with members of the phyla Proteobacteria  is consistent with results from other marine and freshwater microbialite communities  . Microbialite alphaproteobacterial specific contigs were assigned to genera of photoheterotrophic , heterotrophic  and nitrogen-fixing  bacteria . Alphaproteobacterial based nitrogen fixation complements cyanobacterial nitrogen fixation in microbialites likely because of cyanobacterial diel cycles  and heterotrophic  bacteria . MetaCyc dissimilatory sulfate pathways were abundant across the different microbialite morphologies in Pavilion Lake . Sulfate-reducing deltaproteobacteria are often found where carbonates precipitate, and are important drivers of the “alkalinity engine,” by pushing the saturation index via increasing alkalinity . Potential electron donors for sulfate reducing deltaproteobacteria in Pavilion Lake microbialites include acetate, lactate, hydrogen, and formate. Whether sulfate reduction helps or hinders carbonate precipitation depends on the electron donor; hydrogen and formate likely promote precipitation, whereas, other organic carbon sources likely lead to dissolution . Pathways for synthesis of cyanoglobin and cyanophycin, as well as copper metabolism, were associated with cyanobacterial mat-builders in all microbialites . Cyanoglobin is a peripheral membrane protein that binds oxygen with high affinity, is highly expressed under low oxygen and could be restricted to some strains of Nostoc sp. and Anabaena sp. , implying the presence of urea metabolism which may be playing a role in precipitation. Gamma and Deltaproteobacteria specific urease beta subunits and urease accessory proteins (UreD/F) have only been identified in the Pavilion Lake microbialite metagenomes. The linkage of urease related genes to Proteobacteria was unexpected due to the strong experimental evidence that Firmicutes (mainly Bacillus sp.) are the dominant taxa contributing urease related genes . These included antibiotic resistance pathways such as beta-lactamases (class A) that were assigned to the Alpha, Beta, and Gamma classes of Proteobacteria . Genes related to antibiotic resistance could be in response to toxic organic molecules produced by cyanobacterial mats . The occurrence of cobalt–zinc–cadmium resistance proteins, efflux pump proteins, phenylmercury acetate degradation, and chromate resistance was similar across morphologies while arsenite oxidation and arsenate reduction pathways were not found at depths deeper than 25 m . Heavy-metal resistance contigs were taxonomically assigned to Alpha, Beta and Gamma classes of Proteobacteria. Pavilion Lake has low levels of zinc (0.01–0.03 mg L-1) and undetectable levels of cobalt, iron, arsenic and cadmium . Arsenic cycling has been suggested to be a prominent feature in ancient microbial mats over 2.7 billion years old  based on genes that are taxonomically assigned to Alpha- and Beta-proteobacteria. Pyruvate, phytol, and chitin fermentation appear to be the main predicted pathways for the generation of primary alcohols . Primary alcohol fermentation has been linked to microbialite dissolution; however, fermentation also provides substrates that fuel dissimilatory sulfate reduction, which can precipitate carbonate  . In contrast, pathways related to carbohydrate metabolism (carbon-related pathways) were ranked second and accounted for ∼9% of the contigs. Among the carbon-related pathways, ∼45% were related to central (TCA cycle) and one-carbon metabolism , while another ∼45% were related to degradation . Only ∼10% of the microbialite-specific contigs were annotated as carbon-fixation related  . Stable-isotope studies suggest that photosynthetic processes are linked to carbonate precipitation in shallow (<25 m) microbialites . Specifically, T4-like phage  and large algal viruses  dominated the viral sequences in the water and were more abundant than in the microbialites and sediments . The low proportion of viral reads in the microbialite data may be biased by the lack of dsDNA viral genomes in the RefSeq database from microbialites compared to water. Viruses in the water appeared to have higher abundances of proteins related to phage structure (tail fibers), phage replication and phage DNA replication .In the cellular fraction from the water, viral sequences represented >1% of reads; whereas, in total DNA extracted from microbialites, viral sequences comprised >0.05% of reads. ANOVA in STAMP based on RefSeq classification confirmed that virus sequences were relatively more abundant in the water than in microbialites or sediments . CRISPR cas genes were associated with the following taxonomic groups: Chloroflexi , Deltaproteobacteria , filamentous cyanobacteria  and Firmicutes  based on tBLASTx (1e-3) analysis. Likewise, more putative genes involved in phage integration and excision occurred in the microbialites than in the nearby environment . Also, CRISPRs were predicted to be associated with key members involved in microbialite formation, such as filamentous cyanobacteria and Myxococcus sp., implying that the microbialite community is under continuous selective pressure from viral infection.An active role for phages in the microbialites is suggested by the higher relative abundances of predicted genes involved with CRISPRs, phage shock and phage excision . Although gene-specific primers targeted to these groups  captured on filters, suggesting that most viral sequences were from infected cells or from viruses attached to particles. It is not uncommon for filters with pore sizes much larger than viruses to contain many viral sequences . The mose groups  failed tcas sequences implies that the Pavilion Lake microbialites are under selection from viral infection. It is likely that the relative abundance of viral sequences has been underestimated because of the lack of representative viral sequences from microbialites in databases.Consistent with reports for other microbialites , relativThis study demonstrates that the microbial community profile and metabolic potential of modern freshwater microbialites in Pavilion Lake are distinct from those in neighboring sediments and water, consistent with previous findings of spatial variation in microbialite systems. These results confirm the notion that the microbialite communities are not being continuously seeded by organisms from the surrounding environment. Our data further suggests a unique microbialite microbial community that encodes a functional guild which is distinctive and likely related to its overall function of carbonate precipitation.Differences among these metagenomes can be attributed to differing selection pressures among environments, with the microbialite community comprised of taxa essential for microbialite growth, as well as opportunists taking advantage of nutrients and the matrix supplied by filamentous cyanobacterial mats. These findings are consistent with photosynthetic influences on carbonate precipitation by filamentous cyanobacteria, with likely contributions by proteobacteria and acidobacteria.Proteobacteria, which are numerically dominant and likely convey resistance to toxins and heavy metals, and may influence carbonate formation through photosynthesis and urea metabolism. This hypothesis is consistent with previous suggestions of heterotrophic contributions to lithification of Pavilion Lake microbialites. Evidence for urealytic metabolism identified here may suggest an important role for this metabolism in carbonate precipitation  suggest that viruses play an important previously unknown role in the microbialite communities in Pavilion Lake.The prevalence of CRISPR-The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Measurement of intra-retinal layer thickness using optical coherence tomography (OCT) has become increasingly prominent in multiple sclerosis (MS) research. Nevertheless, the approaches used for determining the mean layer thicknesses vary greatly. Insufficient data exist on the reliability of different thickness estimates, which is crucial for their application in clinical studies. This study addresses this lack by evaluating the repeatability of different thickness estimates.Studies that used intra-retinal layer segmentation of macular OCT scans in patients with MS were retrieved from PubMed. To investigate the repeatability of previously applied layer estimation approaches, we generated datasets of repeating measurements of 15 healthy subjects and 13 multiple sclerosis patients using two OCT devices . We calculated each thickness estimate in each repeated session and analyzed repeatability using intra-class correlation coefficients and coefficients of repeatability.We identified 27 articles, eleven of them used the Spectralis SD-OCT, nine Cirrus HD-OCT, two studies used both devices and two studies applied RTVue-100. Topcon OCT-1000, Stratus OCT and a research device were used in one study each. In the studies that used the Spectralis, ten different thickness estimates were identified, while thickness estimates of the Cirrus OCT were based on two different scan settings. In the simulation dataset, thickness estimates averaging larger areas showed an excellent repeatability for all retinal layers except the outer plexiform layer (OPL).Given the good reliability, the thickness estimate of the 6mm-diameter area around the fovea should be favored when OCT is used in clinical research. Assessment of the OPL was weak in general and needs further investigation before OPL thickness can be used as a reliable parameter. The tecse ratio .Today’s SD-OCT devices allow high-resolution, 3D imaging of retinal tissue. They expand the use of OCT from macroscopic qualitative interpretation towards quantitative applications. One emerging field for this quantitative OCT is the measurement of neurodegeneration and neuroinflammation in neurological disorders. As part of the central nervous system, the retina is an excellent site for monitoring neurological pathologies, from well-known and economically significant diseases like multiple sclerosis (MS)  or AlzheRecently, quantitative OCT imaging of the retina has shifted from the peripapillary retinal nerve fiber layer (RNFL) thickness as a marker of retinal neurodegeneration to distinct macular intra-retinal layer thicknesses. However, quantification of intra-retinal layer thickness requires the segmentation of OCT scans into the respective layers. Currently, this task is performed either manually, by experienced graders, semi-automatically, with manual inspectors correcting automated results, or fully automatically. Moreover, the algorithms used to automate the segmentation have been developed independently by the device manufacturers. Consequently, several post-processing algorithms exist that aim to meet this requirement as built-in or add-on software, but the lack of standardization between these limits their prospects for diagnosis and disease monitoring. Until this lack is redressed, OCT, promising as it may be, cannot be used as a reliable marker for neurodegeneration in either therapeutic trials or clinical care, as previously suggested .layer thickness estimates need to be standardized and investigated for reliability and comparability [The necessary reliability standards can be met if two current deficiencies are redressed: Firstly, the current algorithms have to be improved to ensure that all techniques discriminate reliably between different layers . Secondlrability . EspeciaAll research articles that included intra-retinal segmentation of macular OCT scans in MS were retrieved from the PubMed database using the following search term: “optical coherence tomography multiple sclerosis” (search performed on 01/24/2014). Only articles written in English that discussed thickness or volume estimates of distinct retinal layers or combined layers were included. Exclusion criteria were the use of retinal segmentation in OCT scans other than macular scans (i.e. scans of the optic nerve head) and if the segmentation was solely performed to determine the thickness of the whole retina .To test the repeatability of different layer thickness estimates under various conditions we generated three datasets: two control cohorts of 15 healthy subjects each underwent three consecutive examinations with Spectralis SD-OCT (measured by MW) or Cirrus HD-OCT (measured by TO) and 13 MS/CIS patients receiving two consecutive examinations with both OCT devices (measured by JM). A detailed demographic overview of the investigated cohorts can be found in Table A in The study was approved by the ethics committee of the Charité–Universitätsmedizin Berlin and was conducted in accordance with the Declaration of Helsinki in its current version and applicable German laws.OCT examinations were performed with the Spectralis SD-OCT  and the Cirrus HD-OCT 4000 . Both eyes of every subject were measured and included in the analysis. We determined five different Spectralis scan designs replicating the settings used in the published studies that met the inclusion criteria . PreviouFor our repeatability datasets all scan protocols were applied with at least 5 minutes between consecutive examinations. The test subjects were asked to leave the room between examinations and the OCT devices were reset to an initial position. All measurements were performed with activated eye tracker, but without the device’s follow-up function.To analyze repeatability of different Spectralis layer thickness estimates, initial intra-retinal segmentation was performed using the Heidelberg Eye Explorer software (Version 6.0.0.2), after which an operator (MW for controls and TO for patients) reviewed the results and occasionally corrected the segmentation lines where necessary (semi-automatic segmentation). Segmentation line correction was performed individually for each scan without using any other scan for reference. The Cirrus volume scans were automatically segmented with the Cirrus review software (6.5.0.772) with no corrections of segmentation lines possible in this software. The mean thicknesses were calculated for the RNFL and the GCIP see  and expoThe thickness estimates for each layer were analyzed using modified Bland-Altman plots . Here, tThe search term “optical coherence tomography multiple sclerosis” yielded 279 articles in PubMed, of which 27 met the inclusion criteria ,12,17–41For Cirrus OCT two different thickness estimates were used and for both automatic segmentation software were applied  as a measure of the precision were highly dependent on the ICC values: with high ICC values (>0.90) we also found very narrow CI widths (<0.1), but CI widths were wider for lower ICC values.In addition to the mixed cohort, were performed distinct analysis of the patient and control cohorts, see To further investigate differences in single retinal layers, Visual review of the Bland-Altman plots for individual thickness estimates and retinal layers showed no systematic errors or outliers (data not shown).Our investigation showed that a wide range of segmentation approaches and layer thickness estimates are currently used in intra-retinal segmentation studies in MS, unfortunately, with equally divergent reliability. As a result, readers should be careful when interpreting published studies using weak thickness estimates, identified by this study.Spectralis SD-OCT and Cirrus HD-OCT are currently the two most common devices used for intra-retinal segmentation in MS. For the Cirrus HD-OCT the segmentation algorithm of the GCIP has been an integral part of the software since version 6.0 and its reproducibility had been investigated elsewhere . Our repStudies in MS patients have revealed pronounced reduction in the ganglion cell layer and this may be the most important target for tracking neurodegeneration at least in MS patients ,39. SincAnother layer of interest is the INL, in which thickening in the area of the macula has been reported in relation to optic neuritis and MS disease activity ,35,36,43per se noise introduced by small changes in the scan window, as with the other layers. The reliability is therefore less dependent on the thickness estimate than on an exact beam placement during measurement. When investigating OPL in clinical or research settings, an optimal OCT scan with exact beam placement is crucial to prevent uncorrectable OPL differences in the raw data. For this reason, the OSCAR-IB quality criteria, particularly criterion B (“beam placement”), should be followed rigorously [The OPL results deserve special mention. Axons in the OPL of photoreceptor nuclei, commonly known as Henle's fiber layer (HFL), run obliquely in the tissue and its OCT appearance is highly dependent on the angle of the OCT’s light beam relative to this layer . The HFLgorously –48.Our study has the following limitations: data acquisition and manual correction was performed by different operators for the study cohorts, but nevertheless results were still very similar in patients and controls. However, in real multicenter settings more influencing factors are possible, like the use of different scanners or software versions or multiple operators. Further studies are necessary to tackle these issues and such studies would require the inclusion of several operators at different centers, which was beyond the scope our study. Furthermore, we did not have access to analyzable data from RT-Vue and Topcon; and the raw data of these devices could behave differently when looking at different thickness estimates. Lastly, the sample size of our study was relatively low and given its exploratory nature we were not able to perform a-priory sample size calculations. However, CI were very narrow for the excellent thickness estimates and we therefore believe that our data still support the notion of excellent repeatability of some thickness estimates and particular layers.Several clinical trials using OCT-derived parameters as primary or secondary study outcome parameters are ongoing or are about to commence. Current and future studies should favor the most reliable thickness estimates over weaker ones. Although the thickness estimates A—E all performed well, the overall performance of estimate A–the mean of all values in the 6mm-diameter ring area centered on and including the fovea–was above average and we strongly encourage clinical researchers, as well as device manufacturers, to rely on this thickness estimate when implementing intra-retinal segmentation measurement across all macular layers. This thickness estimate should also serve as the basis for acquiring normative data from healthy controls to aid comparability between different devices and to detect measurements deviant from healthy subjects. If a proven reliable thickness estimate is not viable within the context of an individual study, scientists should explain how they selected the alternative methods and quantify the reliability of these.S1 Data(ZIP)Click here for additional data file.S1 FileThis file contains overviews of the study cohorts (Table A), the scientific articles and OCT scan settings used for retinal OCT segmentation in patients with multiple sclerosis (Table B) and the applied segmentation methods used in the retrieved articles (Table C).(DOCX)Click here for additional data file.


African American women with SLE experience faster progression and worse consequences of disease compared with their White counterparts. This study sought to examine whether self-reported routine experiences of discrimination, as a source of psychosocial stress, is associated with disease damage among African American women with SLE.Participants were 578 African American women in the Georgians Organized Against Lupus study, a population-based cohort of SLE patients in Atlanta, GA, USA. Disease damage was assessed using the Self-Administered Brief Index of Lupus Damage (SA-BILD), a validated, patient-reported measure of organ damage since the onset of SLE. Discrimination was assessed using the Everyday Discrimination Scale, a widely used measure of routine experiences of unfair treatment. Ordinary least-squares regression analyses were used to examine the outcome of SA-BILD score by the primary predictors: unfair treatment, racial discrimination attribution, and their interaction, controlling for age and years since SLE diagnosis.n = 258 compared with n = 146). Age  and years since diagnosis  were significantly correlated with SLE damage. Reports of unfair treatment and making an attribution to racial discrimination were not significantly associated with SLE damage. In multivariable regression analyses controlling for age and years since diagnosis, we found a significant interaction between unfair treatment and attributions to racial discrimination . Greater unfair treatment attributed to nonracial causes was associated with higher SA-BILD score, whereas unfair treatment attributed to race showed an inverse association , and the mean years since initial diagnosis was 13.6 years (SD = 9.3). The mean unfair treatment score was 1.92 (SD = 0.95), indicating that on average participants reported experiencing each of the forms of unfair treatment approximately once a year. A total of 159 participants (27.6%) reported not experiencing any unfair treatment. Among participants reporting any unfair treatment, most did not make an attribution of racial discrimination (This study highlights the role that social stressors have in contributing to the progression of SLE and is the first to examine whether unfair treatment and racial discrimination are associated with disease damage among African American women with SLE. Consistent with findings from studies on discrimination and other health outcomes, these results suggest more complex, interactive rather than direct associations with SLE damage, with differential relationships being found between those who attributed unfair treatment primarily to racial discrimination versus those who did not.


Late gadolinium enhancement (LGE) cardiac MRI is the clinical gold standard for non-invasive characterization of myocardial scar . The LGEThe TI scout sequence is an inversion prepared cine sequence with TrueFISP readout. Severe off-resonance from an ICD produces severe banding artifacts in TrueFISP images. In addition, in IR sequences, severe off-resonance can prevent inversion of spins in the myocardium, giving rise to hyper-intensity artifacts ,3. RecenFigure We have modified the TI scout sequence by implementing a wideband inversion pulse and FLASH readout. Phantom results and patient studies demonstrate that the modified sequence has none of the image artifacts that occur with the standard sequence, and produces a reliable estimation of inversion time. We expect that the new TI scout sequence will lead to improved application of LGE MRI in patients with implantable cardiac devices.NIH 1R21 HL118533.