The optimal treatment for thoracolumbar fractures (TLF) without neurological deficit remains controversial. Majority of the systematic reviews and meta-analyses have evaluated open operative approaches but have yet to compare the outcomes of minimally invasive percutaneous pedicle fixation (MIPPF) versus non-operative treatment. A retrospective cohort study was performed to compare clinical and radiological outcomes between MIPPF and conservative groups for TLF AO Type A1 to Type B2 during a 2-year follow-up period. Pre-operative plain and CT films were evaluated and decision made for short segment (non-fusion) MIPPF. Patients who refused operation were treated conservatively with three months of body cast, brace, or corset. MIPPF group showed earlier Visual Analog Score(VAS) improvement at six months post-injury (0 vs 6.0- p<0.001), as well as better functional and radiological outcomes (p<0.050) at final follow-up. Progressions of regional kyphosis (RK) were noted in both groups but there was no significant difference within and between them(p>0.050). MIPPF as a method of internal bracing can be pursued in the treatment of TLF, with larger future cohorts and RCTs being called for to support and explore new findings. MIPPF confers advantages of posterior musculature preservation with less blood loss, shorter operative time, lower infection risk, less postoperative pain, shorter rehabilitation time and shorter hospital stay compared to that of open approach12. Studies have demonstrated efficacy of minimal invasive percutaneous pedicle fixation as an option of management for AO Type A and Type B thoracolumbar fractures in neurologically intact patients6. Several systematic reviews and meta-analyses of non-operative versus operative treatment for TLF have been conducted9. Majority of the studies included in these reviews had evaluated all types of operative approaches, namely posterior, anterior, or combined antero-posterrior. The literature is however, lacking in the comparison of - MIPPF operative approach with that of conservative treatment. We therefore performed a retrospective cohort study to evaluate the clinical and radiological outcomes of operative treatment using MIPPF technique with that of non-operative treatment for TLF AO Type A1 to Type B2 in neurologically intact patients.Thoracolumbar junction (T11 to L2) is the most common vertebral segment susceptible to fracture due to the transition of the relatively immobile rib-bearing thoracic vertebrae articulating with the more mobile lumbar vertebral segment. The optimal treatment for thoracolumbar fractures (TLF) without neurological deficit remains controversial. A vast number of TLF can be treated with traditional open approach of pedicle screw fixation. The advent of minimally invasive percutaneous pedicle screw fixation (MIPPF), originally aimed at treating degenerative spine pathologies, is increasingly being used in the last few decades to treat certain types of TLFOperative registry search for all MIPPF TLF cases performed from January 2009 March 2011 as well as patients' registry and database search for all TLF (T11 to L2) cases  treated conservatively during the same period were carried out. Pre-operative plain radiographic imaging of antero-posterior and lateral views and Computed Tomography (CT) films were evaluated - based on the AO Classification and decision made for short segment (non-fusion) percutaneous pedicle fixation (one level above and one level below the fractured vertebra). Patients who had refused operation were treated conservatively with bed rest, analgesia and immobilization. Methods of immobilization in the conservative group consisted of either body cast, corset or thoracolumbar orthosis for three months. Inclusion criteria were: traumatic spine fracture, thoracolumbar level fracture involving T11 to L2 levels, AO Spine Fracture Classification Type A1.3, A3.3 and B2.3, non-pathological fracture, age 18 and above, patient without neurological deficit. Exclusion criteria were: pathological fracture, fracture involving more than one vertebral level at thoracolumbar region, patients with neurological deficit. Two spinal systems had been used: Depuy Viper II and Medtronic Sextant II percutaneous cannulated screws. All the MIPPF surgery were performed by a single surgeon.Patients' clinic follow-up records were examined for both clinical and radiological outcomes that were being monitored throughout regular outpatient follow-up. (1) pain, measured using Visual Analog Score (VAS)- ; (2) function and quality of life, measured using validated indices of Short-form (SF) 36 health survey . Radiological outcomes: (1) Cobb's angle of regional kyphosis measured in degrees (2) percentage of vertebral height compression in relation to that of adjacent one vertebral level cranial and caudal to the fracture level.Data entry, cleaning and analyses were done using SPSS Version 18.0 for Windows. Descriptive statistics were used for baseline characteristics of both groups. Results for the outcomes of study were analyzed with non-parametric T-test Mann-Whitney test, Wilcoxon Signed Rank test, Spearman's Correlation test and reported with corresponding 95% confidence interval and p values. Less than 0.05 of p value was considered statistically significant. Clinical outcomes of VAS and SF 36 Physical and Mental Component Summary Scores were reported at six weeks, six months, 12 months and 24 months follow-up interval while radiological outcomes were evaluated at six weeks and at final follow-up. Approval for this study was obtained from the ethical committee of the institutional review board.There was a total of 19 cases, 10 of which had undergone short segment MIPPF and nine patients had opted for conservative treatment. The average follow-up for MIPPF and conservative groups were 25 weeks and 28 weeks respectively. The MIPPF group demonstrated better clinical outcome- (p <0.050) compared to the conservative group throughout the 2-year follow-up period - . Additio2 = 0.239. However the relationship was not statistically significant (p = 0.535). There was a negative correlation between MCS Score and degree of regional kyphosis, r2 = -0.208 in the MIS group but there was no statistical significant difference, (p = 0.564)  .16, hence the lack of consensus in its decision-making. We are aware that in recent years, MIPPF is increasingly being used in the treatment of TLF as a method of internal bracing. However, evidence in the literatures on the efficacies of this method of treatment for spine trauma is limited to a small case series and case reports21. Small comparison studies between open surgical techniques and percutaneous pedicle screw placement have demonstrated that the latter has the same advantages as the former which include restoration of sagittal alignment and stabilization of fractures but with less of the morbidities associated with open exposures such as high intra-operative blood loss, prolonged duration of surgery, increased infection rates and paraspinal muscle denervation or injury12. As for the non-operative group, proponents advocating this option pointed out the morbidities and complications associated with open stabilization approaches, hence the unnecessary subjection of patients to operation in view of absence of significant differences in the outcome being observed in the long term9. While some patients may benefit from conservative approach, the requirement for prolonged bedrest, compliance issues with cast or brace may not be feasible for the majority of patients. Moreover, there are potential complications of progressive spinal deformity, persistence of pain and occasional neurological compromise3. Therefore, in our study, we were interested in evaluating whether MIPPF would be a good option in combating the challenges of conservative management in the treatment of neurologically intact TLF without subjecting patients to the potential morbidities of traditional open approaches.Systematic reviews and meta-analysis on the treatment of TLF with and/or without neurological injury have not shown sufficient clear evidence demonstrating its optimal treatment modalitieset al9 in their Cochrane Systematic review concluded that there was no statistically significant difference in the pain and function related outcome two years or more post-injury between operative and non-operative treatments for TLF without neurological deficit. Sonali et al7 in their meta-analysis on non-operative versus operative treatment for TLF without neurological deficit also demonstrated no differences in VAS pain score and functional outcome between the two groups at last follow-up. The functional outcome of SF- 36 PCS Scores and MCS Scores in our study showed significant differences (p < 0.050) in the MIPPF group throughout the follow-up period. At the last follow-up, the MIPPF group demonstrated better functional outcome compared to those in the conservative group (PCS: 97.50 (3.9) vs 72.50 (30.94)- p <0.001; MCS: 96.73 (3.31) vs 88.25 (7.81)- p =0.002). The contrasting results of our study compared to those mentioned in the reviews may be attributed to the difference in methods of surgical approaches in the operative groups, i.e. MIPPF versus traditional open approaches. Their meta-analysis7included all operative groups regardless of the types of surgical treatment, namely traditional open posterior, anterior, or combined approaches with or without fusion. To our - knowledge, there is no comparative study in the literature between the conservative group and the relatively recent MIPPF used specifically as a method of internal immobilization in the treatment of TLF, which thereby poses to us as limitation to allow for comparison of our current results.Our study showed statistically significant difference in the VAS scoring of MIPPF group at baseline until last follow-up with earlier improvement being observed as well at six months post-injury (0 vs 6.0- p < 0.001). Yi et al22 reported that the kyphotic progression appears to occur in the initial post-injury period, with relative stabilization of kyphosis noted within 12 to 18 months. In our study, we did not evaluate the radiological parameters at 12 months post-injury. Chaichana et al23 in their literature review reported that the MIPPF technique is of advantage to expeditiously immobilize a thoracolumbar fracture with minimal or no kyphosis. Our further statistical analysis however did not show significant differences in the progression of RK within and between the two groups. Moreover, our study showed that there was no significant association between degree of kyphosis and functional outcomes in both groups. This is consistent with findings from other studies31. The VH and RK would be expected to progress more in the group of osteoporotic patients. Association test between age and vertebral height compression showed a positive correlation in the conservative group but was not of statistical difference when there was no relationship demonstrated in the MIPPF group. These findings have implications in justifying the clinical importance of kyphosis as a common outcome measure in other studies34.Although there were significant differences in radiological outcome (RK and VH) between MIPPF and conservative group with the former demonstrating better outcome both at baseline and at last follow-up (MIS: 8.00 (8.00) vs 23.00 (16.00) (p = 0.014), progression of kyphosis was noted to occur in both groups. Reid 35. Technical issues in MIPPF surgery call for a learning curve with the prerequisite of ample surgical experience in traditional open posterior approach where direct visualization of anatomical landmarks may assist in accuracy of screw placement and reduce the likelihood of pedicle wall violation thereby shortening operating time and complications of general anaesthesia. Patient selection is paramount in that the risks and benefits should be weighed out particularly for those with underlying co-morbidities that may potentially preclude them from undergoing surgery under general anaesthesia while bearing in mind the possible complications if treated conservatively. The cost of MIPPF implants remains a point of debate. Within the proper context, one can perhaps rationalize that earlier functional improvement and the return to work in the MIPPF group could be overall cost-effective. However, future cost-analysis studies in this regard may be worthwhile.In this study, there was no complication documented in the MIPPF group. No cases of infection, loss of fixation, fracture non-union and neurological compromise were reported. In the conservative group, compliance rate to body cast and brace posed - limitations. We are unable to comment on whether there was any significant difference in the clinical and radiological outcomes using different methods of external bracing and casting. We acknowledge the limitations of our study in being a retrospective study with small sample size. We could not perform subgroup analysis based on fracture types due to small sample size. Points to note in the MIPPF group are the issues of cumulative exposures to radiation to both surgeons and patients alike, as well as the costs of cannulated percutaneous screw systems. The drawback of radiation exposure in fluoroscopic-assisted MIPPF is undoubtedly a concern not to be taken lightly. Navigation system or computer-assisted surgery system available today may be advantages in reducing irradiation time and exposureOur study showed that minimally invasive percutaneous pedicle screw fixation as a method of internal bracing can be pursued in the treatment of thoracolumbar fractures. Comparison between minimally invasive operative group and conservative group demonstrated better functional and radiological outcomes with statistically significant differences in the MIPPF operative group at baseline and throughout the follow-up period. In the light of this, future larger comparative cohorts and randomized controlled studies are called for to support our study results and perhaps explore new findings that will be of greater benefit in contributing to the decision-making for treatment of thoracolumbar fractures.


Metformin lowers diabetic blood glucose primarily by reducing hepatic gluconeogenesis and increasing peripheral glucose uptake. However, possible effects by metformin on beta-cell function are incompletely understood. We speculated that metformin might positively influence insulin secretion through impacting the beta-cell nitric oxide synthase (NOS)-NO system, a negative modulator of glucose-stimulated insulin release. In short-time incubations with isolated murine islets either glibenclamide or high glucose augmented insulin release associated with increased NO production from both neural and inducible NOS. Metformin addition suppressed the augmented NO generation coinciding with amplified insulin release. Islet culturing with glibenclamide or high glucose revealed pronounced fluorescence of inducible NOS in the beta-cells being abolished by metformin co-culturing. These findings were reflected in medium nitrite-nitrate levels. A glucose challenge following islet culturing with glibenclamide or high glucose revealed markedly impaired insulin response. Metformin co-culturing restored this response. Culturing murine islets and human islets from controls and type 2 diabetics with high glucose or high glucose + glibenclamide induced a pronounced decrease of cell viability being remarkably restored by metformin co-culturing. We show here, that imposed overactivity of the beta-cell NOS-NO system by glibenclamide or high glucose leads to insulin secretory dysfunction and reduced cell viability and also, importantly, that these effects are relieved by metformin inhibiting beta-cell NO overproduction from both neural and inducible NOS thus ameliorating a concealed negative influence by NO induced by sulfonylurea treatment and/or high glucose levels. This double-edged effect of glibenclamide on the beta-cellsuggests sulfonylurea monotherapy in type 2 diabetes being avoided. Although sulfonylureas also have efficient glucose-lowering properties, such a treatment has evolved, because clinical studies have revealed that sulfonylurea monotherapy has a pronounced tendency to fail over time i as well as the recently described stimulation of Epac2A [Since the pattern of ncNOS and iNOS activities found in the presence of glibenclamide at 7 mmol/l glucose is almost identical to the pattern found at high glucose (20 mmol/l), it seems conceivable to conclude that glibenclamide, and most probably also other sulfonylureas, has the ability to induce a previously less understood beta-cell dysfunction through imposing overactivity of the NOS-NO system in the background of its well-known stimulation of insulin release through closure of the beta-cell Kf Epac2A . Thus glIt should be recalled that strongly increased levels of islet NO production in both rodent and human islets have been reported to have an inhibitory influence on both nonoxidative and oxidative metabolism of glucose leading to a reduced ATP/ADP ratio and a reduced insulin secretion ,32. ExceThe mechanisms underlying the beneficial effects of metformin on the beta-cell have for long been unclear. Metformin has been suggested to be a positive modulator of glucose-stimulated insulin release through activation of AMP-activated protein kinase (AMPK), but recent studies concluded that AMPK in fact is a negative modulator of the release process . PreviouAs previously mentioned, metformin is recommended as a first-line agent in the treatment of impaired glucose tolerance and type 2 diabetes mainly because of its ability to reduce hepatic glucose production and increase insulin-stimulated glucose uptake in muscle and fat ,2,5. TheFrom the present data we conclude that the islet NOS-NO system is a more important regulator of insulin secretory mechanisms than previously anticipated. Imposed and persisting overstimulation of this system by glibenclamide and/or high glucose will result in an excessive production of NO with the capability of inhibiting crucial steps along the insulin secretory signalling pathways and in the long term eventually lead to a reduced beta-cell viability. The negative influence of the concealed dysfunctional NO effects induced by glibenclamide or high glucose on beta-cell activity can be counteracted, relieved and to a significant part explained by the ability of metformin to downregulate this imposed overactivity of the NOS-NO system both in short- and long-time perspective. Hence, in this new perspective, the beta-cell failure after long term sulfonylurea treatment is now more understandable and the present experimental results, from the point of view of the beta-cell, strongly suggest sulfonylurea monotherapy in type 2 diabetes being avoided.


Sec and discuss the local and global rearrangements of the ribosome in response to correct and incorrect aa-tRNAs. We present the mechanisms of GTPase activation and GTP hydrolysis of EF-Tu and SelB and summarize what is known about the accommodation of aa-tRNA on the ribosome after its release from the elongation factor. We show how ribosome dynamics ensures high selectivity for the cognate aa-tRNA and suggest that conformational fluctuations, induced fit and kinetic discrimination play major roles in maintaining the speed and fidelity of translation.Elongation factors Tu (EF-Tu) and SelB are translational GTPases that deliver aminoacyl-tRNAs (aa-tRNAs) to the ribosome. In each canonical round of translation elongation, aa-tRNAs, assisted by EF-Tu, decode mRNA codons and insert the respective amino acid into the growing peptide chain. Stop codons usually lead to translation termination; however, in special cases UGA codons are recoded to selenocysteine (Sec) with the help of SelB. Recruitment of EF-Tu and SelB together with their respective aa-tRNAs to the ribosome is a multistep process. In this review, we summarize recent progress in understanding the role of ribosome dynamics in aa-tRNA selection. We describe the path to correct codon recognition by canonical elongator aa-tRNA and Sec-tRNAThis article is part of the themed issue ‘Perspectives on the ribosome’. Fromchniques ,37. Fromchniques ,40. No sruitment   conformation  at the h12/S4/S5 region and at the intersubunit bridge B8 [Trp accelerate the rates of GTP hydrolysis independently of codon–anticodon pairing [Trp accomplishes this by increasing tRNA flexibility, whereas G24A tRNATrp allows the formation of an additional hydrogen bond that stabilizes the distortion [One remaining question is why the mismatches in the codon–anticodon complex impede GTPase activation. The rate of GTP hydrolysis increases by three to seven orders of magnitude in response to the correct codon–anticodon interaction in the decoding centre ,39,41,65ridge B8  are in lridge B8 . However pairing . Mutatiostortion . Howeverstortion ,67; the 6.+ ion coordinated by Asp21 has a small stimulatory effect. Upon binding to the ribosome the mechanism of GTPase reaction changes [Ka of His84 is shifted upwards so that the sidechain is positively charged at neutral pH [2+ ion is not resolved in EF-Tu, but is clearly seen in the ribosome-bound structures of SelB and EF-G  [−1) and SelB (10 s−1).EF-Tu and SelB have a nucleotide binding pocket which is conserved in the majority of translational GTPases . Two uni changes –72. The  changes ,73. Ribo changes ,74. The  changes ,75; the  changes . Both ob changes –72. The utral pH –72. The ctively) ,76,77. I7.Accommodation comprises the movement of aa-tRNA after its release from EF-Tu into the peptidyl transferase centre on the LSU . The tRNThe biological significance of tRNA accommodation is in providing the mechanism for the aa-tRNA proofreading . When thSimilarly to the initial selection step, the proofreading rates are defined by the codon–anticodon base pairing. The interactions between the cognate codon–anticodon complex and the ribosome elements that resulted in global SSU domain closure during the initial selection step are likely maintained during proofreading, because structures of the complexes in the A/T and A/A states indicate identical interactions in the decoding centre. By contrast, it is less clear whether the rearrangements induced by the near-cognate codon–anticodon complexes are the same or different at the initial selection and proofreading stages; ribosome mutations can differentially affect the aa-tRNA binding stability at the initial selection and proofreading stages . Those n8.Sec to the ribosome demonstrates that we can now tackle such complex heterogeneous dynamic systems by both structural and biophysical approaches. The ribosome is a dynamic molecular machine; understanding how spontaneous and induced fluctuations of the ribosome and other translational players are rectified into rapid and accurate translation will answer fundamental questions about the movement and selectivity of molecular ensembles.Structural and kinetic work of the past two decades suggested an important contribution of ribosome dynamics, induced fit and kinetic discrimination in the mechanism of decoding and recoding. Recent breakthrough developments in cryo-EM now allow us to follow the ribosome as it selects aa-tRNA in each round of translation. We can now see more clearly how translational GTPases work and how the ribosome discriminates between the correct and incorrect substrates. Knowing the rate constants of decoding by different aa-tRNAs provides the numbers to build predictive models for translation and to explain the existence of natural translational pauses. The mechanism of stop-codon recoding by Sec emerges in great molecular detail. Understanding the structural basis of tRNA selection will require visualizing the analogous sequence of decoding intermediates for EF-Tu-mediated aa-tRNA delivery. Recent work on deciphering the binding pathway of SelB–Sec-tRNA


The Dishevelled/EGL-10/Pleckstrin (DEP) domain-containing (DEPDC) proteins have seven members. However, whether this superfamily can be distinguished from other proteins based only on the amino acid sequences, remains unknown. Here, we describe a computational method to segregate DEPDCs and non-DEPDCs. First, we examined the Pfam numbers of the known DEPDCs and used the longest sequences for each Pfam to construct a phylogenetic tree. Subsequently, we extracted 188-dimensional (188D) and 20D features of DEPDCs and non-DEPDCs and classified them with random forest classifier. We also mined the motifs of human DEPDCs to find the related domains. Finally, we designed experimental verification methods of human DEPDC expression at the mRNA level in hepatocellular carcinoma (HCC) and adjacent normal tissues. The phylogenetic analysis showed that the DEPDCs superfamily can be divided into three clusters. Moreover, the 188D and 20D features can both be used to effectively distinguish the two protein types. Motif analysis revealed that the DEP and RhoGAP domain was common in human DEPDCs, human HCC and the adjacent tissues that widely expressed DEPDCs. However, their regulation was not identical. In conclusion, we successfully constructed a binary classifier for DEPDCs and experimentally verified their expression in human HCC tissues. Drosophila)Caenorhabditis elegans)mammals)45The Dishevelled  occurrs via an evolutionarily conserved signaling pathway that involves the DEPDC protein LET-99 . This concept suggests that DEPDC1 participates in the anti-tubulin drug-induced apoptotic cell death pathway17DEPDC1 is a highly conserved protein that was first found to over-express in bladder cancer cellsDEPDC2 is broadly expressed in human and zebrafish2426DEPDC3 has no related literature in PubMed, but it is also known as the G-protein-coupled receptor 155 (Gpr155), which is conserved among mammals and may be a candidate gene for type 2 diabetes in mouse modelsDEPDC4 is also a signaling molecule involved in G-protein-coupled receptor (GPCR) signaling pathways. The gene sequences of this protein were disrupted by rearrangement in the gibbon genome during mapping around the breakpoint regions, as compared with the human genomeDEPDC5 is a protein that is strongly associated with various of familial epilepsiesDEPDC6 is also known as the DEPDC mTOR-interacting (DEPTOR) protein, which can only be found in vertebrates. mTOR is a conserved serine/threonine-protein kinase forming mTOR complex [mTORC] 1 and mTORC248Three years ago, DEPDC7 was called LOC91614 and relatively unknown; however, our group has given close attention to this gene and studied it for several years. Our previous studies showed the high level of DEPDC7 differential expression in HCC tissues and hepatoma cell lines; the gene is closely correlated with the proliferation, migration, and invasion capacity as verified by RNA interference researchesMachine learning, developed from computational learning and pattern recognition theory, makes computers able to learn without being explicitly programmed, and it has been widely used to devise prediction modelsBased on the sequence and other physicochemical properties of each protein, we discriminated and predicted the DEPDC proteins from non-DEPDC proteins with a machine learning algorithm by extracting the 188D and 20D feature vectors and constructing a binary classifier for this purpose in this study. Subsequently, we searched for the main motifs of human DEPDC proteins, which are related to their functional domains. Finally, we performed experimental verification of human DEPDC gene expression with qRT-PCR in HCC and adjacent normal tissues.Based on the neighbor-joining algorithm and bootstrap method for phylogeny test, the number of bootstrap replicates was set to 500, and the tree was out-grouped with the “Root On Midpoint” optionWe obtained data on the 188D and 20D feature vectors from the positive and negative groups for import into the Weka explorer. The results showed that the correctly classified rates were 96.2% and 93.2%, whereas the ROC area reached 0.995 and 0.983. The confusion matrix is shown as Our phylogenetic analyses with MAFFT revealed that the eight human DEPDC members can be divided into three main groups . Group IHuman DEPDCs were broadly expressed in HCC. Most genes showed significant differential expression . The expThe DEPDC protein family includes seven members, which contain the DEP domain as their common characteristic; these proteins can mediate protein-to-protein interaction and membrane targeting of signal molecules51859In this study, we performed binary-class classification of the DEPDC and non-DEPDC family. First, a phylogenetic tree was built from all the longest positive Pfam-containing sequences and discovered that the DEPDC family can be divided into three clusters. Based on protein sequence and isual-chemical characteristics, we extracted the 188D and 20D feature vectors on the positive and negative datasets by machine learning, as predicted by RF classifier. We obtained relatively high effects on the correctly classified rates and the AUC value. Similarly, the Sn, Sp, Acc, and MCC values reached relatively superior results. To the best of our knowledge, we are the first to categorize the DEPDC family; thus, no corresponding pre-existing data is available for comparison. Third, the MEME Suite is suitable for screening common sequence motifs from a set of amino acid sequencesFinally, we also used experimental methods to verify the gene expression of the human DEPDC family in HCC and adjacent normal tissues. From the qRT-PCR results, we observed that human DEPDCs were widely expressed in the cancer tissue. However, direction of the expression was not identical. Our experiment showed that DEPDC1 expression was not affected between the two tissues. DEPDC1B was up-regulated in HCC, whereas the other six genes (DEPDC2- DEPDC7) were down-regulated in HCC. This trend may indicate their different functional correlation and great variation. Further study on the regulation and function of these genes with HCC or other related cancers is needed.In conclusion, we have successfully constructed a binary-class model algorithm to split the DEPDCs with non-DEPDCs, and the 160 positive Pfam-containing members can be differentiated into three main clusters. The eight human DEPDCs can be divided into three groups from molecular phylogenetic analyses and the conserved DEP and RhoGAP domains were discovered by MEME tools. Finally, we verified the eight human DEPDCs mRNA expression with experiment in human HCC tissues.www.uniprot.org)http://cd-hit.org) for merging the sequence similaritiesThe primary sequences of DEPDC proteins and the control Pfam proteins (as FASTA files) were retrieved from the Universal Protein Resource (UniProt) database (All positive DEPDCs were applied to extract their corresponding protein families (returned Pfam number) from the Uniprot “Family & Domains” section. After excluding the identical and redundant entries, we acquired 229 unique Pfam numbers (the names begin with PF). To construct a phylogenetic tree6768To fully uncover the important information hidden in protein sequences, we constructed two feature extraction algorithms: the 188-dimensional(188D) and the simplified 20D feature vectors. First, we extracted the feature vectors from the positive and negative protein sequence dataset by a previously developed novel machine learning method7273http://www.cs.waikato.ac.nz/ml/weka/), which is a machine learning workbench. In weka, we filtered the vector data with the synthetic minority over-sampling technique(SMOTE)77788081828384After the above mentioned process, the resulting feature vectors are imported into weka (Sn), specificity (Sp), accuracy (Acc) and Matthew’s correlation coefficient (MCC). The formulas of these parameters are868788To measure the performance quality of the statistical classification more intuitively in machine learning, we calculated four common parameters for evaluating the classifier: sensitivity (TP(true positive) indicates the number of true DEPDCs that are correctly predicted, TN(true negative) indicates the number of true non-DEPDCs that are correctly predicted, FP is the number of true non-DEPDCs that are predicted to be DEPDCs, and FN is the number of true DEPDCs that are predicted to be non-DEPDCs.where http://www.ebi.ac.uk/Tools/msa/mafft/) to generate a phylogenetic tree. MAFFT has been significantly improved in accuracy and reduced CPU time as compared with CLUSTALW; the algorithm is also faster than T-COFFEE90http://meme-suite.org/, 4.11.2 version) was performed for conserved motif analysis. A motif can be assumed as a conserved sequence pattern that repeatedly occurs in a group of related sequences. MEME is a powerful motif-based sequence analysis suite, which can promptly discover novel, ungapped motifs by integrating various sequence analysis tools for proteins, DNA and RNABefore motif searching, the eight human DEPDCs  were implemented by MSA and forwarded to the MAFFT server , as recommended by the manufacturer. Real-time PCR was performed on the Step-OneTM Real-Time PCR system (Applied Biosystems) with the SYBER Green qPCR Supermix (Roche) under universal thermal cycling parameters . The comparative Ct method was used to quantify gene expression−ΔCt.The primers were designed by the PrimerQuest tool of IDT, which is freely available at The nonparametric Mann–Whitney U-testThe study is approved by the ethics committee of the Fuzhou Dongfang Hospital and the experimentation is conducted in accordance with the Declaration of Helsinki and Good Clinical Practice (GCP).How to cite this article: Liao, Z. et al. Identification of DEP domain-containing proteins by a machine learning method and experimental analysis of their expression in human HCC tissues. Sci. Rep.6, 39655; doi: 10.1038/srep39655 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


In the setting of an acute myocardial infarction (AMI), although the most common etiology of a left ventricular (LV) mass identified on multimodality cardiovascular imaging is a thrombus, other possibilities including a vegetation or tumor should be entertained within the differential diagnosis.We describe a case of a 43-year-old Caucasian female post AMI diagnosed with a mid-cavitary mass within the LV. Although echocardiography and cardiovascular MRI (CMR) suggested that the mass was a thrombus, given the context of the recent AMI, exploration and surgical excision was completed by the surgeon due to the potential for the mass to embolize.The final diagnosis of a papillary fibroelastoma was unique due to its unusual location and large size within the LV cavity. This unique case demonstrates shortcomings of multimodality cardiac imaging in the diagnosis of an atypical mass and the importance of obtaining tissue when clinically safe and feasible. Primary tumors of the heart are infrequent with an overall incidence of 0.02% based on autopsy series . With thIn the setting of an acute myocardial infarction (AMI), a LV mass is likely to represent a thrombus. Approximately 7% of patients with an anterior AMI develop a LV thrombus , 3, whicWe describe a case of a 43-year-old female post AMI diagnosed with a mid-cavitary mass within the LV. Despite the use of multimodality cardiovascular imaging, she required surgical excision for the rare diagnosis of a papillary fibroelastoma.A 43-year-old Caucasian woman with type II diabetes, hypertension, dyslipidemia, and pre-existing ischemic heart disease (IHD), presented with a non ST-segment elevation myocardial infarction (NSTEMI). Coronary angiography demonstrated multi-vessel disease with severe in-stent restenosis of her proximal left anterior descending (LAD) and significant obstructive lesions in the proximal right coronary artery (RCA) and proximal obtuse marginal (OM) artery. Given the multi-vessel disease with two layers of stent and aggressive nature of her coronary artery disease, the patient was accepted for in-hospital coronary artery bypass grafting (CABG). A preoperative transthoracic echocardiogram (TTE) confirmed a mass 23 × 18 mm) within the LV cavity  are uncommon, benign, avascular cardiac tumors. With an estimated frequency as high as 0.33% on autopsy series, PFEs constitute up to 7% of all primary cardiac tumors . PFEs arEchocardiography is the most readily and cost effective imaging modality available to assess a LV mass. It provides basic information on mass morphology, mobility, position, and attachment. Contrast echocardiography using Definity allows additional classification based on vascularity . DespiteOur case presents a unique challenge to pre-operative diagnosis. Although echocardiography and CMR suggested that the mass was a thrombus, given the context of the recent anterior MI, exploration and surgical excision was completed by the surgeon due to the potential for the mass to embolize. The final diagnosis of a PFE was unique due to its unusual location and large size within the LV cavity. This unique case demonstrates shortcomings of multimodality cardiac imaging in the diagnosis of an atypical mass and the importance of obtaining tissue when clinically safe and feasible.


The aim of this study was to evaluate the Vickers surface micro-hardness and the depth of cure of two bulk-fill resin composites and one incremental-fill resin composite.Two Bulk-fill dental resin composites  and an incremental-fill dental resin composite  were used. Sixty cylindrical specimens of 4 mm thickness were prepared using split Teflon moulds. Specimens were divided into six groups (n=10) according to the type of the material used and according to the insertion technique applied . Prepared specimens were stored dry in complete darkness at 37°C for 24 hours. All specimens were tested for their Vickers surface micro-hardness, on their top and bottom surfaces. The depth of cure of the tested specimens was assessed by calculating the hardness ratio for each specimen. The Vickers surface micro-hardness and depth of cure data were analyzed for normality using Kolmogorov-Smirnov and Shapiro-Wilk tests. Independent sample-t test was used to compare between two groups while One-way ANOVA was used to compare between more than two groups.P<0.0001). X-tra Fil recorded the highest mean Vickers micro-hardness value (94.05±1.05). Bulk-fill dental resin composites X-tra Fil and Sonic-Fill showed 0.980±0.005 and 0.921±0.020 depth of cure values (bottom/top hardness ratio) respectively while Z250 XT recorded 0.776±0.141.Significant difference in the Vickers surface micro-hardness and depth of cure values was demonstrated among the tested materials (X-tra Fil showed highest Vickers surface micro-hardness values on both top and bottom surfaces, whether inserted in increments or bulk. Both bulk-fill resin composites showed higher depth of cure for both insertion techniques. Key words:Depth of cure, Vickers surface micro-hardness, bulk-fill resin composite, insertion techniques. Restoring prepared dental cavities with light-curing resin composites has been regarded as the gold standard. However, to apply and cure the resin composite in successive increments of limited thickness has shown to be time-consuming and increase the risk of incorporating oral fluids within the increments,  which adRecently, many dental practitioners prefer using more time-saving restorative procedures. Dental resin composite manufacturers, with a vision to simplify these procedures; have introduced a new category of dental resin composites, the so-called “bulk-fill” Materials,  that canTherefore; the objective of this study was to gain more insight about bulk-fill resin composite materials by analyzing the effect of different insertion techniques on the Vickers surface micro-hardness and the depth of cure of two different bulk-fill dental resin composite materials.This study has been approved by ethics committee. Two commercially available bulk-fill dental resin composites and one incremental-fill dental resin composite were used in the study. Materials’ description, manufacturers and composition were listed in -Study design and specimen grouping.Sixty cylindrical specimens were prepared and assigned for the depth of cure and Vickers surface micro-hardness evaluation. Specimens were divided into six groups (n=10/group), representing the resin composite materials used in the study (two bulk-fill resin composites: Sonic-FillTM2 [Sonic-Fill] and X-tra Fil [X-tra Fil]); one incremental-fill resin composite: FiltekTM Z250 XT [Z250 XT]; and the two insertion techniques .-Specimen preparation:Split Teflon moulds of 4 mm diameter and 4 mm thickness were used to prepare the specimens. Each mould was encircled by a copper ring to provide stabilization during manipulation of the materials.The moulds were first mounted on the top of a microscope slide and a clear Mylar strip, and then each mould was filled with one of the three resin composites  according to the insertion techniques investigated in the study. For the bulk insertion technique; each mould was filled in a 4 mm single increment. While for the incremental insertion technique; the resin composite materials were inserted into the moulds in two successive increments (2 mm thickness each).The first resin composite increment was photo-polymerized using LED light curing unit  with a light intensity output of 1000 mW/cm2 for 20 seconds as recommended by the manufacturers of the control resin composite material used in the study ; then the second resin composite increment was inserted and packed over the previously photo-polymerized increment till the mould was slightly over-packed. The top surface of each mould was then covered using a second clear Mylar strip to avoid creation of the oxygen inhibited layer. A glass slide and a load of 1 kg was constantly held in place on the top of the second clear Mylar strip for 30 seconds to ensure consistent packing of the specimens and to obtain flat surface .The top surface of each prepared specimen was photo-polymerized using the light curing unit for 20 seconds. The guidance tip of the light curing unit was centered in 90° angle to the top surface of the specimen and kept in a direct contact with the second Mylar strip. Light intensity output of the light curing unit was monitored using Demetron radiometer device   and thenTop surface of each specimen was identified with a dark marker pen. Specimens were stored dry in tightly sealed containers and in complete darkness condition at 37°C for 24 hours to prevent the ambient light from causing post light-curing polymerization, (-Vickers surface micro-hardness testing:Sixty specimens (n=10) were tested using Vickers micro-hardness testing machine  . Three r-Depth of cure of the resin composites:The depth of cure of the tested resin composites was assessed by calculating the hardness ratio for each specimen. It was calculated by dividing the mean Vickers hardness number (VHN) of the bottom surface by the mean (VHN) of the top surface for each specimen according to the equation:  Depth of-Statistical analysisP≤0.05. Statistical analysis was performed with IBM® SPSS® Statistics Version 20 for Windows.Mean and standard deviation values were calculated for each group in each test. Data were explored for normality using Kolmogorov-Smirnov and Shapiro-Wilk tests. Independent sample-t test was used to compare between two groups while One-way ANOVA was used to compare between more than two groups. The significance level was set at P=0.001. The top surface group of X-tra Fil bulk-fill resin composite when used in bulk insertion technique recorded the highest mean micro-hardness value (95.16±0.44). However, the bottom surface group of Z250 XT recorded the lowest mean micro-hardness value when used in bulk insertion technique (57.16±0.69).Means and standard deviations of Vickers surface micro-hardness for the evaluated groups were represented in p=0.0001. The highest mean depth of cure value was recorded for X-tra Fil bulk-fill resin composite when inserted incrementally (0.983±0.001); while the lowest value was recorded for Z250 XT resin composite when inserted in bulk.Mean and standard deviation values of the depth of cure of the tested materials were represented in et al.  and then each increment was photo-polymerized only from the topside in a way to mimic the actual clinical situation. This would eventually mean less resin composite matrix polymerization and, accordingly, a larger role of the filler particle type and percentage (content) in the material behavior .It is expected for any resin composite material that its top surface micro-hardness value to differ from the values recorded from the bottom surface, due to the difference in the monomer reactivity and filler/matrix refractive index mismatch .In the present study, the mean micro-hardness values recorded on the bottom surfaces of Z250 XT were significantly decreased compared to the values recorded on the top surface; however, for X-tra Fil and Sonic-Fill bulk-fill resin composite materials; the values of the micro-hardness of the bottom surfaces were not statistically different from values registered for the top surfaces. The surface micro-hardness of resin composites was proved to be affected by increment thickness of the used resin composite material . In our The significant decrease in the surface micro-hardness of the 4 mm bulk inserted specimens of Z250 XT in this study were in agreement with a previous study  which reThe significant difference between the surface micro-hardness values of X-tra Fil and Sonic-Fill bulk-fill resin composite materials might be attributed to the difference in their monomer viscosity. It was reported that the flowable bulk fill resin composites showed lower surface micro-hardness values than the condensable bulk-fill resin composites . Sonic-FMoreover, there are other parameters that might be responsible for the difference of surface micro-hardness values among the different tested materials including, filler particles morphology and distribution,  particleOn the contrary, Z250 XT failed to fulfill this requirement when inserted in bulk. The higher depth of cure in bulk-fill resin composites could be owed to their higher translucency, as well as to their modified resin monomers and the recent photo-initiator systems ,15. It wSome studies have shown that the translucency was decreased when the amount of the reinforcing filler particles was increased,  while itThis was confirmed by results of the current study where Z250 XT showed significantly lower depth of cure than X-tra Fil and Sonic-Fill. There is some other factor that might explain the differences in the depth of cure among the different tested resin composite materials; which is the resin monomer properties. Moreover; the viscosity of the resin monomer and the flexibility of its compositional chemical structure might influence the depth of cure of the resin composite material. In the current study, X-tra Fil showed significant higher depth of cure than that of the other tested materials. This might be due to the difference in the chemical composition of their organic resin matrix. The organic resin matrix of X-tra Fil is composed of Bis-GMA, UDMA, and TEGDMA. It was stated that the ultimate degree of monomer conversion of the different resin composite systems, regarding their compositional resin monomers, increases in the subsequent arrangement: Bis-GMA<Bis-EMA<UDMA<TEGDMA .Bis-GMA has a strong intra-molecular hydrogen bonding of its hydroxyl groups, in order that it is considered the most viscous and least flexible monomer among the other compositional dental resin monomers. UDMA has a hydrogen bond between its amine and carbonyl groups, and it is also a viscous resin monomer.Nevertheless, the viscosity of UDMA is much lower in relation to that of Bis-GMA due to its weaker hydrogen bond.Additionally, amine groups presence in the urethane structure of UDMA monomer is considered to be responsible for the distinguishing chain transfer reactions that offer an alternative trail for the continuance of polymerization; therefore resulting in a promoted resin monomer conversion . This miWhen incremental insertion was used, a significantly higher depth of cure was noted compared to bulk insertion. Such results were confirmed by a previous study and might be related to the fact that specimens prepared with incremental packing received more total energy than those inserted in bulk .X-tra Fil bulk-fill resin composite material has showed the highest Vickers surface micro-harness values on both top and bottom surfaces of the tested specimens, whether inserted in increments or in bulk. Both bulk-fill resin composites (X-tra Fil and Sonic-Fill) showed higher depth of cure regarding both insertion techniques.


The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) is a genome editing tool that has recently caught enormous attention due to its novelty, feasibility, and affordability. This system naturally functions as a defense mechanism in bacteria and has been repurposed as an RNA-guided DNA editing tool. Unlike zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 takes advantage of an RNA-guided DNA endonuclease enzyme, Cas9, which is able to generate double-strand breaks (DSBs) at specific genomic locations. It triggers cellular endogenous DNA repair pathways, contributing to the generation of desired modifications in the genome. The ability of the system to precisely disrupt DNA sequences has opened up new avenues in our understanding of amyotrophic lateral sclerosis (ALS) pathogenesis and the development of new therapeutic approaches. In this review, we discuss the current knowledge of the principles and limitations of the CRISPR/Cas9 system, as well as strategies to improve these limitations. Furthermore, we summarize novel approaches of engaging the CRISPR/Cas9 system in establishing an adequate model of neurodegenerative disease and in the treatment of SOD1-linked forms of ALS. We also highlight possible applications of this system in the therapy of ALS, both the inherited type as well as ALS of sporadic origin. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) is a novel genome editing tool that has recently revolutionized the field of human genetic engineering. This system naturally serves as an adaptive defense mechanism in bacteria and archaea against foreign nucleic acids such as plasmids and bacteriophages . The CRIEscherichia coli. This stretch of DNA consists of short direct repeats separated by short unique sequences. It was found later that CRISPR/Cas machinery functions analogously in bacteria and archaea to the adaptive immune system in vertebrates, providing a genetic library (memory) of phages and plasmids that previously have invaded the bacteria. Resistance against foreign nucleic acids after the subsequent infection is assured due to the enzymatic activity of the Cas proteins [The discovery of the CRISPR loci began in 1987 when Ishino et al.  first reCRISPR/Cas-mediated immunity acts in three steps: adaptation, expression with maturation, and interference. In the first adaptive phase, bacteria or archaea incorporate short foreign sequences (protospacers) into the host genome generating repeat-spacer motifs. In the next phase, transcription of the repeat-spacer sequences yields precursor CRISPR RNAs (pre-crRNAs) that after maturation become crRNA molecules, which are able to recognize and target invading genetic material in the interference step. Due to the activity of Cas proteins, the invading nucleic acid can be degraded ,13 Figu.Streptococcus thermophilus and is able to specifically cleave double-stranded DNA [Streptococcus pyogenes requires 5′-NGG-3′ sequences adjacent to the target locus [Streptococcus pyogenes can be directed by two RNAs: CRISPR RNA (crRNA) and trans-activating RNA (tracrRNA) constituting crucial components of the type II CRISPR/Cas system. Moreover, dual-tracrRNA:crRNA can be successfully replaced by an engineered single guide RNA (sgRNA) to simplify the task of programming Cas9 [Among the three existing types of CRISPR/Cas systems, the type II system involving Cas9 proteins has proven to be the most promising genome engineering tool ,14. The nded DNA . The sitet locus . Jinek eThe double-strand breaks (DSBs) induced by the Cas9-sgRNA complex are repaired by either of two major distinct pathways: nonhomologous end joining (NHEJ) or homology-directed repair (HDR). Both of them are able to operate in almost every cell type and organism  Figure . NHEJ isThe simplicity of the CRISPR/Cas9 system encourages the exploration of its potential to generate animal models or to treat severe and incurable human diseases. However, potential users need to consider several issues that limit the feasibility of this system.The guide RNA of the system does not need to fully match the target sequence for its recognition and cleavage . This chThe other concern is the delivery of sgRNA and Cas9. Different strategies involve viral and non-viral vectors to introduce components of the system. Lentiviral (LV) or adenoviral vectors (AV) are common and effective tools used as delivery systems for sequence-specific sgRNA and Cas9 nucleases ,28,29. NAlthough the immunogenicity of viral vectors used for in vivo delivery has been taken into consideration, little is known about the ability of the bacterial Cas9 protein to trigger an immune response in humans . CertainThe above-mentioned hurdles of the CRISPR/Cas9 system reveal its imperfection. Nevertheless, much effort has already been made to increase the specificity and efficiency of the system. Although the risk of off-target mutagenesis dramatically undermines the CRISPR/Cas9 system, various approaches have been undertaken to avoid undesirable mutations and increase on-target efficiency. The potential off-target effects can be diminished from the beginning of the experiment through the selection of an appropriate target site and the designing of the sgRNA. It has been revealed that the GC (guanine–cytosine) content of sgRNA has an influence on the rate of off-targets .One of the approaches to elicit a higher specificity of this technique can be a simultaneous expression of two guide RNAs targeting opposite DNA strands . Paired Additionally, the off-target cleavage can be significantly diminished without loss of on-target efficiency by using truncated guide RNAs (tru-gRNAs). The truncated guide RNA fragments recognize shorter target sequences of less than 20 nucleotides . This apFokI nuclease (fCas9). Two monomers of fCas9 are able to modify target DNA with at least a 4-fold higher specificity than paired Cas9 nickases (Cas9n), retaining the efficiency comparable to that of paired Cas9n [fCas9 and the truncation of gRNAs. Such a combination provides highly efficient and specific genome modification.Another strategy to improve the on-target mutagenesis with less off-target effects is the fusion of catalytically inactive Cas9 to ed Cas9n . Furthered Cas9n  concatenCas9 gene and sgRNA in plasmids carries the risk of a prolonged expression of RGENs, which increases the number of off-target events. Thus, in vivo therapy requires a “hit and run” strategy that depends on the introduction of Cas9 protein instead of the Cas9 gene. This solution assures immediate induction of mutations such as insertions or deletions at target sites and rapid degradation of RGEN ribonucleoproteins leading to off-target reduction [The application of the CRISPR/Cas9 system for therapeutic purposes requires a safe and efficient method of provisioning the components. Delivering the eduction .The establishment of a good animal model for neurodegenerative diseases that faithfully simulates human illness requires mimicry of the symptoms as well as developing pertinent neuropathological features. A successfully-generated animal model provides a significant contribution to the understanding of disease pathophysiology and helps to develop therapeutic and preventative strategies. Genetic conditioning of neurodegenerative diseases makes it possible for them to be imitated in animal models by using genome engineering tools. Even though the generation of genetically modified animal models has been abundant in the last two decades, most of them referred to small animals such as invertebrates or rodents ,45,46. CThe development of the CRISPR/Cas9 tool has opened the door for the advancement of new model systems in neuroscience. These models include in vivo animal models as well as stem cell-derived models for studying “a disease in a dish”. The traditional strategies are based on microinjection of genetically modified embryonic stem cells into blastocysts for germline transmission. Unfortunately, such approaches require much time and effort and might be inefficient in introducing genome modification. Additionally, the traditional methods are not applicable for generating large animal models. The CRISPR/Cas9 system has many advantages in modeling neurodegenerative diseases and, therefore, might help to avoid the above-mentioned problems of traditional technologies .The application of the CRISPR/Cas9 system as a novel tool for modeling neurodegenerative diseases has been facilitated due to its ability to generate mutations in endogenous genes. On the other hand, traditional methods lead to the expression of mutant genes under exogenous promoters and, therefore, are not useful for modeling the diseases. Furthermore, the CRISPR/Cas9 system is able to introduce mutations in one or two alleles in order to mimic heterozygous or homozygous knock-outs of a given gene. Targeting both alleles results in a null mutation in the model organism. This is significantly beneficial for generating large animal models since the generation of homozygous mutants by mating two heterozygous mutants is highly time consuming. Additionally, the disruption of both alleles in female animals enables the complete loss of function of a given gene when the disease is linked to the X chromosome .parkin, DJ-1, and PINK1 genes by multiple sgRNAs co-injected into pronuclear embryos of Bama miniature pigs. The genetically modified piglets remained healthy with a normal growth rate. Although the 10-month-old mutant pigs did not display typical symptoms of Parkinson’s disease, the influence of the triple-gene targeted modification was confirmed on a molecular level. Immunofluorescence and Western blot analysis revealed the absence of DJ-1 protein expression in the fibroblasts, confirming the inactivation of the DJ-1 gene on both alleles. The expression of the parkin and the PINK1 gene determined by real-time RT-PCR was also significantly decreased in the fibroblasts of genetically modified pigs when compared with wild-type cells. Moreover, despite the high number of sgRNAs that were employed, the trio-based whole-genome sequencing analysis demonstrated a low incidence of off-target effects [The CRISPR/Cas9 system has been recently used for engineering the pig genome in order to generate a human Parkinson’s disease model. In this study, three different genomic loci were targeted including  effects . The resThe CRISPR/Cas9 system was also successfully employed to target monkey genomes. The co-injection of Cas9 mRNA and sgRNAs into one-cell-stage embryos enabled concurrent modification of two target genes (Ppar-γ and Rag1) in one step. Fortunately, the off-target mutagenesis was not identified by a comprehensive analysis. Taken together, the study confirmed the reliability of the system for simultaneous disruption of two genes that led to the generation of a genetically modified cynomolgus monkey .SOD1) gene. The remaining familial ALS cases are associated with mutations in various genes among which the most common are mutations in the 43-kDa TAR DNA-binding protein (TARDBP) gene [FUS) gene [In the context of ALS, CRISPR/Cas9 seems to be an attractive tool since ALS is a rapidly progressive, fatal neurodegenerative disease that can be the result of a combination of genetic and environmental factors . InheritBP) gene  or in thUS) gene . C9orf72US) gene .TARDBP and FUS genes using the CRISPR/Cas9 system to develop an ALS model in zebrafish. Although CRISPR/Cas9-mediated NHEJ led to the generation of INDELs, it failed to create single nucleotide substitutions. Nevertheless, co-injection of single-stranded oligodeoxynucleotide donor templates (ssODN) containing point mutations with gRNA and Cas9 mRNA resulted in HDR integration of the two missense mutations . Thus, this approach has proven that homology-directed repair (HDR) following CRISPR/Cas9 enables the generation of knock-in lines that imitate disease-causing point mutations in humans using zebrafish TARDBPA379T (TARDBPA382T) and FUSR536H (FUSR521H). Since CRISPR/Cas9-mediated NHEJ has limited potential to model phenotypes, HDR is recommended for that purpose. Unfortunately, CRISPR/Cas9-mediated HDR can be problematic due to its low efficiency and therefore various novel approaches have been developed to increase efficiency, likely leading to a better integration of the donor template. It might seem that the expression of a human gene with diminished homology to the ortholog in a phylogenetically distant species can be insufficient and too biased to reflect the human disease. Fortunately, the CRISPR/Cas9-mediated breaks and HDR ssODN template knock-in successfully avoids this restriction by editing the endogenous gene. Secondly, this novel technique saves time and labor since genomic integration in zebrafish can be evaluated within two days [Taking into consideration the genetic background of familial ALS cases, several studies have been conducted to develop transgenic ALS models, among which the recently discovered CRISPR/Cas9 system holds great promise for a simple, feasible, and inexpensive method for generating model systems. Armstrong et al.  have rectwo days .C9orf72 gene has been recently discovered as a major cause of frontotemporal lobar degeneration (FTLD) with amyotrophic lateral sclerosis (ALS) [hSOD1G93A ALS mouse model. The genes encoding several neurotrophic factors are being investigated in the context of ALS due to their therapeutic potential in neurodegenerative diseases. The gene encoding insulin-like growth factor 1 (IGF1) was knocked-down in this mouse model in order to observe and verify the processes that are influenced by IGF-1 [Additionally, CRISPR technology has been used to develop pathogenic variants of ALS in mice. By taking advantage of the system’s ability to create several patient-specific mutations in genes coding different domains of a single protein, it is now possible to recapitulate the disease caused by various mutations . The CRIis (ALS) . Furtherby IGF-1 .2+ homeostasis and decreased cell survival corresponding to a diminished rate of anti-apoptotic protein Bcl-2. Overall, it was delineated that iPSC-derived motor neurons are valuable cellular models of neurodegenerative disorders and are a novel genome engineering tool that can facilitate discoveries.This versatile genome editing tool has recently received attention for its potential in cellular models of ALS. The applicability of the CRISPR/Cas9 system for studying a “disease in a dish” that employs the patient’s own derived induced pluripotent stem cells (iPSCs) was highlighted. Mutihac et al.  from theG93A mouse model have revealed that silencing of the mutant SOD1 gene using both interfering RNA (iRNA) [The fatal outcome of ALS accelerates the urgent need for the discovery of novel therapeutic approaches that may lead to the development of a cure for this devastating disease. Conventional therapies are focused on symptoms and are not able to eliminate the pathology underlying the clinical manifestation. The potential of gene therapy to permanently affect the intracellular processes is promising for the treatment of incurable neurodegenerative diseases. Genome engineering tools are used to target other inherited movement disorders such as Parkinson’s disease both in preclinical studies  and in cA (iRNA)  and artiA (iRNA)  delayed A (iRNA)  demonstrC9orf72 gene, the common cause of familial ALS. One of these approaches involves the CRISPR/Cas9 gene editing tool to remove the mutant gene through enzymatic cleavage; the other strategy is focused on delivering RNA in order to reduce C9orf72 expression [The application of the CRISPR/Cas9 system in the therapy of ALS currently has a dual purpose: gene therapy for inherited ALS and the expression of genes encoding neurotrophic factors (NTFs). Recently, the University of Massachusetts Medical School received a grant to develop two gene therapies to target the mutant pression .NTFs genes would be another possibility [The second approach of using CRIPSR/Cas9 technology in ALS therapy is based on the meaningful potential of neurotrophic factors to exert neuroprotective effects on damaged motor neurons in both familial and sporadic ALS cases. Although NTFs emerged as promising candidates for ALS treatment in the early 1990s, initial treatments proved to be ineffective especially due to delivery issues . More cosibility .The CRISPR/Cas9 system makes genome engineering technology feasible for application in many fields including human diseases. Current research on this innovative genome editing tool and strategies to improve its drawbacks has laid the groundwork for future clinical work in neurodegenerative diseases. It has opened new avenues for studying the complexity of neurodegeneration in both in vivo and in vitro model systems. The ability of the system to establish large animal models that recapitulate specific human diseases improves our understanding of complex mechanisms involved in disease pathology. Lessons learned from modeling neurodegenerative diseases in large animals using CRISPR/Cas9 give a confirmation that despite several limitations of the system, this new technology is able to effectively modify animal genomes with the absence of off-target mutagenesis. Targeting multiple genes in the same cell by CRISPR/Cas9 technology enables the study of synergistic effects due to the loss of crucial genes. Although in the context of ALS research the CRISPR/Cas9 system is still rather limited to generating small animal models or improving the in vitro models, results obtained from studies mentioned in this review may facilitate the field of modeling ALS.Moreover, the feasibility of the CRISPR/Cas9 technology to treat ALS revealed recently in mice gives enormous hope to patients suffering from ALS. Although such approaches are currently restricted to reducing the expression of mutant proteins in inherited cases of ALS in mice, the system might bring breakthroughs in both the familial and the sporadic origin of the disease in the future.The possibilities of CRISPR-mediated genome editing summarized in this review both in modeling and possible treatment of ALS have begun a new era in ALS research and the CRISPR/Cas9 system has emerged as a feasible tool to achieve what has seemed impossible for decades.


Autoimmune and allergic diseases occur when an individual mounts an inappropriate immune response to a self-antigen or an innocuous environmental antigen. This triggers a pathogenic T-cell response resulting in damage to specific tissues and organs. In type 1 diabetes (T1D), this manifests as destruction of the insulin-secreting β cells, resulting in a life-long dependency on recombinant insulin. Modulation of the pathogenic T-cell response with antigen-specific peptide immunotherapy offers the potential to restore the immune homeostasis and prevent further tissue destruction. Recent clinical advances with peptide therapy approaches in both T1D and other diseases are beginning to show encouraging results. New technologies targeting the peptides to specific cell types are also moving from pre-clinical development to the clinic. While many challenges remain in clinical development, not least selection of the optimal dose and dosing frequency, this is clearly becoming a very active field of drug development. Type 1 diabetes (T1D) is an organ-specific autoimmune disease. Auto-reactive T cells attack the insulin-producing beta (β) cells of the pancreatic islets, leading to a loss of endogenous insulin production and subsequent impaired glucose metabolism. Management of the disease requires daily administration of exogenous insulin and frequent monitoring of blood glucose levels. While there have been significant advances in recombinant human insulins and technologies both to deliver insulin and monitor blood glucose levels, many patients do not achieve optimal glycemic control. This is particularly apparent in children and young adults, in whom blood glucose control, measured by glycated hemoglobin (HbA1c) levels, is typically poor, reaching greater than 8% in the majority when the desired levels are below 7.5% . This may in part be explained by a large placebo effect that was observed and the transition to field-based studies, which inherently introduce greater variability. An ongoing clinical study focusing on the phenotype of the allergen-specific T cells following administration of Cat-PAD will confirm whether peptide therapy can modulate the pathogenic T-cell response .Clinical proof of concept that peptide therapy can restore immune homeostasis has been championed by studies of allergic disease. Cat-PAD is a peptide cocktail consisting of seven peptides from Feld1, the major cat allergen. A recent clinical study has shown that a short course of intra-dermal treatment with these peptides resulted in a clinically meaningful reduction in rhino-conjunctivitis symptoms, 2 years post-treatment . DisappoClinical studies with peptides derived from auto-antigens for the treatment of autoimmune disease are also now emerging. A phase IIa study with a peptide immunotherapy treatment for relapsing remitting multiple sclerosis demonstrated statistically significant reductions in total and new T1 Gadolinium enhancing brain lesions (representing sites of inflammation and damage) measured using magnetic resonance imaging during treatment. This therapy consists of a cocktail of four peptides derived from myelin basic protein.For peptide therapy to be successful in treating autoimmune or allergic disease, it is essential that the major auto-antigens responsible for driving the disease are known. Using the presence of auto-antibodies to the same antigens as a guide increases the probability that appropriate antigens have been selected. The ability to detect antigen-specific T cells is also important not only to aid the initial epitope discovery of disease-relevant peptides but also to track modulation of the T-cell phenotype following clinical administration.ex vivo  derived from the insulin B9-23 epitope (NBI-6024) showed some initial promise . ModulatIn parallel to the clinical development of NBI-6024, a natural peptide sequence derived from proinsulin was also under evaluation. An initial phase 1 safety study in patients with long-standing diabetes and low c-peptide levels (<200 pmol/L) compared three monthly intra-dermal injections of 10 versus 100 µg of the C19-A3 peptide . The pepex vivo stimulation and enhanced expression of the canonical regulatory T cell transcription factor FoxP3 on peripherally generated, adaptive regulatory T cells post-therapy. These findings, linking clinical and immunological outcome provide an encouraging platform on which to build strategies for biomarkers of peptide immunotherapy.Subsequently, a second clinical study with the single proinsulin peptide was conducted in newly diagnosed patients and the results have recently been reported . In thisClinicalTrials.gov: NCT02620332). This phase Ib tolerability and safety study evaluating monthly intra-dermal injections of a cocktail of multiple peptides should provide new insights when data become available in 2018.The clinical studies described thus far, both in T1D and the other indications, have focused on either a single peptide to modulate the pathogenic T cell response or multiple peptides from the same antigen. A different approach, using multiple peptides from two different auto-antigens linked to the pathogenesis of T1D, is currently being evaluated as a treatment for new-onset T1D patients .A slightly different peptide therapy approach to those previously described is also under clinical evaluation. The approach is to take a natural peptide derived from one of the major auto-antigens linked to T1D and modify it slightly to improve its affinity . These mThe peptide therapy approaches leading the way in clinical development for both T1D and other autoimmune diseases are relatively simple. The peptides are administered either intra-dermally or sub-cutaneously in the absence of any additional substantial modifications. Whether these approaches are sufficient to bring significant clinical benefit in large phase III clinical studies remains to be seen, but certainly, the promise peptide therapy holds for restoration of immune regulation has led a number of pharmaceutical companies to develop novel and innovative ways to target the peptides in a more specific manner for potentially enhanced efficacy. These include targeting of peptides to the erythrocyte , encapsuex vivo assays that selectively stimulate antigen-specific cells (in vivo data from mouse studies that have clearly demonstrated expansion of an IL-10 secreting, regulatory T-cell population following sub-cutaneous administration of peptide (While peptide therapy for T1D is clearly entering an exciting phase of drug development, there are still a number of unanswered questions and challenges, not least whether the approach will bring real clinical benefit. Selecting the optimal dose of peptide and optimal dosing interval is one key challenge. This is in part because it is difficult to study both the kinetics of peptide uptake and the bio-distribution of peptides. Access to better immunological tools to track the antigen-specific T cells will undoubtedly help our understanding; this includes further development of both tetramers and ic cells . The abi peptide . Translavia the generation of IL-10 producing regulatory T cells, the Foxp3+ regulatory T cell, or a combination of both? It will also be important to understand the longevity of the effect and whether this is dependent on the phenotype of the regulatory T cell. It may be that the different routes of administration of peptides and the newly emerging targeted technologies mentioned previously lead to the generation of different populations of regulatory T cells. It is not inconceivable to think that different diseases may be better treated by induction of one particular population of regulatory T cell versus another. In addition, different stages of progression within the same disease may be more amenable to treatment by one subset of regulatory T cell over another. This may be particularly true for diseases where there is significant epitope spreading. In this situation, one might envisage early in disease using a peptide treatment that induces the Foxp3+ regulatory T cell and later in the disease pathogenesis, using a peptide treatment that induces the IL-10 secreting regulatory T cell.Once the technologies and assays have been further refined so that tracking of antigen-specific T cells, both pathogenic and regulatory, can be accomplished, additional questions can start to be addressed. Importantly, degrees of clinical efficacy can begin to be correlated with both the frequency of regulatory T-cell expansion and the phenotype of the expanded regulatory T cells. Is better efficacy achieved via inhibition of T-cell co-stimulatory receptors such as CD40L, OX40, and ICOS or via engagement of negative regulators such as PD-1 and TIGIT. Modulation of the cytokine milieu may also synergize with the efficacy of peptide therapy. This could be achieved via direct inhibition of the proinflammatory cytokines secreted by the pathogenic T cell or inhibition of cytokines responsible for maturation of the APC.One must also acknowledge that administration of peptide therapy alone may not be sufficient to prevent disease in an inflammatory setting. In this setting, the peptide therapy may indeed be inducing a population of regulatory T cells, but the inflammatory environment may also be driving a pathogenic T-cell population, which is not adequately suppressed by the regulatory cells. Therapies that directly inhibit the pathogenic T cell while sparing the regulatory T cell would be optimal under these circumstances. These could include therapies that target the APC to prevent full activation, achieved for example using antibodies against CD40. Alternatively, therapies that inhibit effector T-cell function directly could be beneficial. This could be achieved either While the challenges are undoubtedly there, peptide therapy does offer a significant opportunity to restore immune regulation in T1D and other autoimmune diseases. Further clinical studies and technological advances will hopefully translate peptide therapy into a safe, effective, and highly specific novel class of therapeutics.ES wrote the review and was involved in designing the figures. MP was involved in designing the figures and supervised the writing and editing of the manuscript.ES is an employee of UCB Pharma Ltd. who are developing peptide immunotherapy for type 1 diabetes. MP is employed by King’s College London, which has a licence agreement in place with UCB Pharma to develop peptide immunotherapy. MP receives research funds and honoraria from UCB.


Several species produce crystals of unusual elongated particle shapes, which break the symmetry of the thermodynamically favoured isometric morphology. Such morphologies are thought to affect domain size and orientation of the internal magnetization. Therefore, they are interesting study objects to develop new synthetic strategies for the morphological control of nanoparticles. We investigate the formation of such irregularly shaped nanomagnets in the species Desulfovibrio magneticus RS-1. In contrast to previously described organisms, this bacterium accumulates iron predominantly as Fe(II) rather than Fe(III) consistent with an alternative oxidative biomineralization route. Further, using high-resolution electron microscopy, we observe an epitaxial relationship between precursor and the final mineral phase supporting the notion of a solid-state transformation pathway. The precursor is likely a green rust previously thought to convert to magnetite only by dissolution and re-precipitation. Our findings represent a novel observation in the interconversion of iron (oxyhydr)oxide materials and suggest that solid-state growth processes could be required to produce irregularly shaped, elongated magnetite nanocrystals.Magnetotactic bacteria are aquatic microorganisms that intracellularly mineralize ferrimagnetic nanoparticles enabling the cells to align with the geomagnetic field. The bacteria produce a magnetic mineral of species-specific phase (magnetite Fe(II)Fe(III) The particle was found in proximity to Fe-P granules and mature magnetosomes  suggesting that granules can transform to magnetite.Over the time course of induced mineralization, we observed several particles by HRTEM with incomplete morphologies b–f suggeetosomes g–i. For etosomes . The chec,d). Of particular interest is a twinned magnetite crystal onto which another phase is attached in evident surface contact oxides and (oxyhydr)oxide phases to the FFTs of the observed micrographic data . While the patterns cannot be unique, the interfacial contact between magnetite and the unknown phase, as well as the angular dependencies between both minerals' lattices indicate an epitaxial relationship between them. Here, epitaxy refers to mutually shared crystal orientations as defined by the two-dimensional lattice at their interface where the atomic positions have to be coherent for both phases. The required coherence in the lattice reduces the list of candidates for which such an overlap is possible. We found two phases for which we could determine such an overlap in the lattice: GR II and ɛ-Fe2O3 is a rare ferric iron oxide phase that is reportedly formed under unusually harsh chemical conditions, for example, by pyrolysis inside heat-treated samples of infiltrated mesoporous silica  direction  consistent with an earlier observation by X-ray diffraction texture analysis .4.D. magneticus RS-1 is preceded by the precipitation of a solid ferrous precursor that differs from previous observations in magnetotactic α-Proteobacteria and other organisms such as the chiton that produce isometric particles (b). This precursor and the intracellular Fe redox chemistry indicate an oxidative magnetite formation pathway. These findings corroborate those on the abundance of redox-regulating proteins in magnetotactic bacteria [We show that biomineralization of magnetite in articles b. This pbacteria . The forD. magneticus RS-1 has four major intracellular iron pools . Iron is most rapidly accumulated in the Fe-P phase that is either purely or largely in the ferrous state . Over time, iron is increasingly present in the other iron pools with a strong preference for ferrous biomolecular Fe-S. Electron microscopy indicates that Fe-P granules can convert to magnetite; however, the wide abundance of the granules at the early stages, but low abundance at late stages in correlation with the observed increasing iron contributions in the Fe-S and ferritin pools suggest that most iron is transferred to those . It has been claimed earlier that iron from the amorphous Fe-P granules either does not contribute to magnetite formation or does so only in minor amounts [figure 8a).Furthermore, our results provide a link between the different intracellular iron pools observed in RS-1:  amounts . Our finDesulfovibrio spp. can produce intracellular polyphosphate granules devoid of iron [D. gigas are large granules (hundreds of nanometres in diameter) and singular [Desulfovibrio spp. may resolve the role of both structures.Note that non-magnetotactic  of iron . This rasingular . In addi5.D. magneticus RS-1 indicates a potential link of the environmental redox conditions of the bacterial habitat and the bacteria's physiology to the applied pathway. Reductive routes could be favoured for bacteria (e.g. magnetospirilla) living under microoxic conditions (where iron is mostly ferric), whereas oxidative routes could dominate for species such as RS-1, living in anoxic conditions (where iron is mostly ferrous). To determine whether the specific cellular iron chemistry of D. magneticus RS-1 and/or the solid-state transformation are linked exclusively to the morphology of the particles or only represent a feature of the cellular Fe physiology, will require the study of further strains that produce anisotropic magnetite particles. We envision this knowledge to enable new strategies for the synthetic production of such magnetic nanoparticles.The observation of a ferrous precursor to magnetite in


High-frequency activity (HFA) is believed to subserve a functional role in cognition, but these patterns are often not accessible to scalp EEG recordings. Intracranial studies provide a unique opportunity to link the all-encompassing range of high-frequency patterns with holistic perception. We tested whether the functional topography of HFAs (up to 250Hz) is related to perceptual decision-making. Human intracortical data were recorded  during an ambiguous object-recognition task. We found a spatial topography of HFAs reflecting processing anterior dorsal and ventral streams, linked to decision independently of the type of processed object/stimulus category. Three distinct regional fingerprints could be identified, with lower gamma frequency patterns (<45Hz) dominating in the anterior semantic ventral object processing and dorsoventral integrating networks and evolving later, during perceptual decision phases, than early sensory posterior patterns (60-250Hz). This suggests that accurate object recognition/perceptual decision-making is related to distinct spatiotemporal signatures in the low gamma frequency range. Gamma-band oscillations have been proposed to play a functional role across a wide range of cognitive domains –4 but thThe seminal proposal that information transfer across neural networks is mediated by a variety of high-gamma frequencies ,15 needsA few intracranial studies have suggested the presence of increased broad and/or narrow band gamma-oscillations during holistic perception. Lachaux et al. 2005 showed an increase of gamma activity between 50 and 150Hz in response to specific Mooney stimuli (two-tone abstract patterns depicting objects), comparing to the baseline. Hermes et al., 2014 reported a spatial distribution of increases in narrow and broadband oscillations with the broadband component being mainly located in early visual areas. Contrary to the frequently claimed stimulus specificity of the narrowband gamma oscillations, both suggested that all classes of visual stimuli induced responses in a broadband frequency range (80-200Hz) in a large portion of the fusiform gyrus and the visual cortex. Moreover, only part of the gamma activity (30-100Hz) is stimulus specific . It is however possible, that in spite of the relative stimulus independence, such patterns may instead reflect general processing demands during perceptual decision-making and object recognition. Here we tested this hypothesis.To test whether activity in the gamma-band represents particular signatures of specific processing modules, we investigated whether these patterns are organized in distinct spatial maps, as a function of the timing of perceptual decision-making. Although a specific functional role of distinct sub-bands has been suggested for different sources in the brain –26, thisThere is still a lot of controversy concerning the spatial organization of those patterns of activity and their functional relevance. While some authors reported that large increases in gamma power  dominateInvasive recordings are often used in the clinical context ,35. TheyWe acquired invasive electrophysiology data from six epileptic patients  of 2x8, 4x8 and 4x5 electrodes were used). The number and placement of the electrodes was determined by the clinical needs of the patients. To avoid movement of subdural electrodes after placement they were stitched to the edge of dura mater and digital photos were taken to confirm position of the electrodes on the 3D anatomical data acquired with MRI and CT.Anatomical MRI, CT data and X-ray images  were acquired before and after subdural electrode placement, respectively. The individual data were then co-registered (using 3DSlicer software 4.3.0 r22408) to precisely localize the positions of the intracranial electrodes . Then, we applied a semi-automatic segmentation to extract electrode positions. The image data of the five subjects with left grids were co-registered to obtain a single map of all electrode locations . The datThe subject was presented with four different categories of Mooney pictures. The categories included Mooney Faces, Mooney Guitars, Scrambled non-face stimuli and inverted Mooney Faces. To make the task easy to understand, for each stimulus, subjects were instructed to report if a face was present  or not , after stimulus offset . Stimulireaction time data were calculated to assess the validity of the responses to the task. All subjects were able to understand and perform the task above chance level. A Wilcoxon statistical test was applied to compare response rates per category (p = 0.05).Behavioral and Then, invasive data were visually assessed by an experienced epileptologist. Electrodes including epileptic spikes or artifacts were marked as bad (~20%). After artifact correction  a common average reference (excluding bad channels) was computed to avoid distributions that are biased by the location of the reference electrode . Data weEEGLAB toolbox [MATLAB v8.1 . The wavelet parameters were: 6 cycles, 250 frequency bins, 600 time points and the analysis frequency range was set to 15 to 500Hz. A notch filter was applied (45-55Hz) to avoid potential line current artifacts. The change between post and pre-stimulus epochs was assessed. Time-frequency data per channel and subject were obtained and a semi-automatic iterative channel clustering procedure was applied based on the similarity of frequency-dependent responses to the stimuli. Briefly, we applied the k-means cluster analysis (MATLAB R2013a) to find the clusters of channels with a similar frequency dependent response (spectrum of the 0-600ms time window). The default squared Euclidean distance measure was used for clustering with input parameters k = 3 and 100 replicates. This clustering approach minimizes the sum, over all clusters, of the point-to-cluster centroid distances. Channels belonging to the same centroid (cluster) were concatenated per condition and analyzed together  decomposition) analysis –45 was p toolbox  and MATLStatistical comparisons were performed per cluster to compare the time–frequency results with the baseline and between stimulus conditions, with the alpha level set to 0.0125. A Wilcoxon rank sum test was carried out per time and frequency bin vs. baseline values.statcond function from EEGLAB was applied using paired t-tests with bootstrapping (2000).Additionally, we computed the spectrum as given by the time-frequency transform to assess the changes during and after stimuli presentation. The group trimmed mean (10% threshold) of the activity spectrum per time-window  was computed  at the sThe resultant P values from the statcond function and Wilcoxon tests were corrected for multiple comparisons using a false discovery rate (FDR) approach (p = 0.0125). Furthermore, to reduce the number of type-1 errors we follow the work by  and corrWe calculated the percentage of correct responses per category. All subjects were able to perform the task (discriminating between face and non-face objects) and could distinguish between face and non-face stimuli above the chance level. Behavioral data in We investigated high frequency activity patterns as recorded with high spatiotemporal resolution using ECoG, during ambiguous perceptual decision making. Intracranial data analysis focused on visual processing regions covered by the occipito, parietal and temporal arrays of electrodes–corresponding to early visual and the dorsal/ventral pathways, which represent an important dichotomy in high level visual processing ,51.We found distinct activity patterns occurring with distinct timing during the ambiguous visual perception decision task across distinct regions in the brain. These patterns did indeed prove to be distinct in frequency and temporal onset of significantly increased activation. These effects occurred up to the upper 250Hz boundary. We performed a semi-automatic cluster analysis see  up to thTo further clarify the relation of the distinct frequency band patterns with perceptual decision, the average envelope of ERSP per conditions was computed for two distinct time-windows. A statistical t-test with bootstrapping (2000) was performed (including FDR p<0.0125 and cluster-based correction for multiple comparisons) to compare the activity during and after stimulus presentation (during decision) per frequency bin. The spectrum per time-window /conditions / cluster are shown in We were able to demonstrate a distinct topographic map of low (< 45 Hz) and high (up to 250 Hz) frequency activation patterns during a perceptual decision making task. The former were present in ventral anterior regions during the decision period and the later dominated early on during visual stimulation. These results are consistent with the notion distinct frequency bands have distinct sources and functional significance ,27,37 anECoG has been widely use to study high frequency oscillations ,40. HerePrevious reports suggested a functional dissociation between low and high gamma sub-bands ,28,52,53We now provide evidence for topographic organization of visual sensory and perceptual processing and establish that these are related to a general object processing mechanism. We used a broad range of new ambiguous stimulus categories   which alThe frequency dependent spatial patterning can be summarized as follows: the low gamma and beta activity spatially involved mainly anterior regions  during perceptual-decision and object recognition while the higher frequencies (up to 250Hz) are increased at posterior areas  early onSome evidence is available pointing to a distinction between sub-bands or differences in broad vs. sharp band patterns across different types of paradigms ,23,27,37Here we found a perceptual-decision related graded posterior-anterior sharpening of frequency band response amplitudes and their respective sources at the cortical surface level. We provided evidence that this occurs at distinct frequencies and as a function of the cognitive moment . In addition, we analyzed every frequency bin in an unbiased manner without any sort of selection. de Pesters et al. 2016 had suggested possible sources of distinct oscillatory patterns within the beta and gamma ranges and we now show a topographic dichotomy with defined functional significance. This is in line with previous EEG/fMRI evidence suggesting that lower frequency patterns are relevant during the decision period, irrespective of the object category. Additionally, we found a spatial map of decreased beta activity located at anterior motor areas. We thus suggest a posterior-anterior topographic pattern reflecting a processing hierarchy, from early visual procession to decision and object recognition.This pattern of temporal and location precedence and dynamics of information processing is consistent with the known hierarchy of visual processing in the anterior dorsal/ventral streams. Given their close relationship to neuronal spiking, this ubiquity of high-frequency patterns, suggests that high-gamma may convey general mechanisms of cortical information transmission , namely In sum, we have found a frequency dependent spatial patterning during object recognition, which is independent of the particular object category, suggesting a general processing mechanism. The perceptual search for an object and ensuing recognition seems to generate distinct spatio-temporal signatures during visual processing and decision. We thus found evidence in favor of the hypothesis that high frequency activity (up to 250 Hz) are spatially and temporally organized in three clusters  as a function of visual processing and perceptual demands.activity. The clear posterior-anterior pattern is consistent with hierarchical processing schemes from posterior to visual anterior dorsal/ventral streams and suggests that such object recognition related patterns are region and local connectivity dependent irrespective of the processed category.To our knowledge, this is the first time that such a functional topographic map is shown in relation to accurate perceptual decision. Furthermore, we show also a time and space dependent  patterning of high frequency gamma S1 FileFigure A in S1 File. Group Average Time-frequency activity per condition. Event-related spectral power (dB) were computed in EEGLAB per subject and condition. This figure summarizes the TF results per condition and cluster of electrodes . The black lines mark the time-frequency blobs that are significantly different from the baseline . The detailed statistical values of the significant TF blobs are reported in Table A in S1 File. The dashed lines mark the start and end of the stimulus presentation. Figure B in S1 File. Time-frequency results for the individual subject 6 . A) Semi-automatic clustering of the spectrum per channel. 2 channel clusters are identified (best sumd = 454.58). B) Time-frequency plots and source locations for the two clusters of channels. Data (dB) are a cluster average for the Mooney faces condition (similar results are found for the other conditions). The dashed lines mark the start and end of stimulus and the black line signal the TF blobs significantly different from the baseline . C) The power envelope of distinct time-windows and clusters of electrodes for the faces condition (mean ± SD). Lower frequencies have higher power after stimulus offset. Horizontal black bar indicate the significant differences . Figure C in S1 File. Locations of the epilepsy related electrodes superimposed in the results image. The ‘red labeled’ electrodes represent the SOZ electrodes across all subjects while the others are the ones reported in our results. Note that there is no overlap because we did not include these ‘bad electrodes’ in the analysis. A normalized time-frequency activity (group average of these SOZ electrodes) locked to the Mooney faces stimuli is also shown. Table A in S1 File. Statistical results. The statistical test results are reported per condition and cluster of electrodes for the two main comparisons. * Wilcoxon rank sum comparison of TF data with the baseline. ** T-test with bootstrap (2000) comparison of the spectrum per time-window (Stim On vs. Decision Period). The max and minimum values of significant blobs are reported (FDR and cluster-based corrected p = 0.0125).(DOC)Click here for additional data file.