N = 163) derived from computerized text-analysis of trauma and control narratives provided within 18 days post-trauma. Trauma narratives with fewer cognitive processing words (indicating less cognitive elaboration), more death-related words (indicating perceived threat to life), and more first-person singular pronouns (indicating self-immersed processing) predicted greater PTSD symptoms at 6 months. These effects were specific to trauma narratives and held after controlling for early PTSD symptom severity and verbal intelligence. When self-report questionnaires of related processes were considered together with the trauma narrative linguistic predictors, use of more first-person singular pronouns remained a significant predictor alongside self-reported mental defeat. Language-based processing markers may complement questionnaire measures in early forecasting of post-trauma adjustment.Identifying early predictors for psychiatric disorders, such as post-traumatic stress disorder (PTSD), is crucial for effective treatment and prevention efforts. Obtaining such predictors is challenging and methodologically limited, for example by individuals' distress, arousal, and reduced introspective ability. We investigated the predictive power of language-based, implicit markers of psychological processes ( There is large heterogeneity in psychological responding following exposure to traumatic events . TraumaA particularly important scientific question therefore is to identify early predictors of adjustment trajectories. To the extent that clinicians can reliably identify who is likely going to do well and who is at a high risk for developing chronic PTSD, limited therapeutic resources can be allocated to where they are most needed. In the context of trauma, identification of predictors at an early stage is an important objective. Survivors often have contact with professional services in the initial aftermath of a trauma, whereas such contact appears much more difficult to establish later, when disorders are chronic, often comorbid and more difficult to treat , 5.Ideally, predictors of later chronic PTSD would be (a) early markers that can facilitate optimization of treatment initiation and resource allocation (b), naturally observable for clinicians—so that assessment burden can be kept to a necessary minimum at a distressing and vulnerable time for trauma survivors, and (c) trauma-specific—so that prediction errors are minimized. Finally, as the validity of self-report questionnaires may be undermined by factors such as high distress, emotional arousal, and limited introspective ability—factors that characterize information processing in the aftermath of a trauma- such markers would ideally be independent of the survivor's explicit self-report and could thus significantly complement such measures.Natural language markers derived from individuals' spontaneous word use have recently received increased scientific attention . Use of Which trauma memory processing styles have been associated with later PTSD? According to cognitive models Participants also completed the National Adult Reading Test , a widecognitive processes  as an index of elaboration and cognitive processing, death-related words  as an index of mental defeat and death salience, negative emotions , as an index of negative emotionality, and first person singular pronouns  as an index of self-immersed processing. To address the question of process specificity, we computed separate word-use variables for the trauma and negative event narratives. Sample excerpts illustrating these four language variables in both types of narratives are provided in the Verbatim transcripts of the narratives were analyzed using Linguistic Inquiry and Word Count , an extData driven processing was assessed as an index of lacking elaboration and cognitive processing with the 8-items data-driven processing subscale from the Cognitive Processing Scale . The scaThe Mental Defeat Scale , an 11-iThe Negative thoughts about the self subscale of the Post-traumatic cognition inventory  was useWe calculated a hierarchical linear regression analysis in order to determine whether trauma-specific linguistic markers predict PTSD symptom severity at 6 months, over and above initial PTSD symptoms, verbal intelligence and non-trauma linguistic variables. From the significant trauma-specific linguistic variables and corresponding self-report questionnaires, we determined the best set of variables to predict PTSD symptom severity using a stepwise linear regression and a forward selection method. All analyses were conducted using SPSS 20.0.Results from the linear regression analyses are shown in Table p < 0.031, alongside self-reported mental defeat, β = −0.52, p < 0.001. No other significant predictor emerged in this analysis. Together, the variables explained 34% of PTSD symptom severity, R = 0.58, F = 31.92, p < 0.001. There were no significant sex differences in magnitude in any of the linguistic predictors under study .In a separate model, we determined the best predictors of later PTSD symptom severity from the three significant trauma narrative linguistic variables  and the corresponding self-report questionnaire scores  using linear regression and a forward selection procedure. First person singular pronouns in trauma narratives significantly predicted PTSD at 6 months, β = 0.17, Linguistic markers of trauma-specific processing indexed in the early aftermath of a trauma uniquely predicted later chronic PTSD symptoms. Less cognitive processing words (as marker of less elaboration), use of more death-related words , use of more first person singular pronouns (as marker of self-immersed processing) assessed in the first 2 weeks after trauma predicted more severe PTSD symptoms at 6 months follow-up. Importantly, these linguistic predictors uniquely emerged from trauma-narrative competing against language markers of general emotional processing derived from non-traumatic, negative event narratives and predicted PTSD over and above established risk factors such as verbal intelligence and initial levels of PTSD symptoms.The use of less cognition words in those with increased PTSD symptoms is in accord with cognitive theories of PTSD and the proposed beneficial nature of elaborative cognitive processing during trauma , 14 and Use of more first-person singular pronouns remained a significant predictor alongside self-reported mental defeat, when corresponding questionnaire measures were included. Assessment of such candidate early linguistic predictors, along with self-report questionnaires is thus one way to capture psychological processes after exposure to trauma and to predict later psychological adjustment. Self-report questionnaires may often provide practically the most efficient way of indexing psychological processing, despite their potential limitations when administered early after of trauma. However, patients routinely report on aspects of their trauma and such narratives could readily be subjected to automatic linguistic analyses as part of routine clinical practice.The current study is not without limitations. Our sample consisted of assault survivors (who suffer a relatively high risk for chronic PTSD) and generalizability to other trauma types needs to be established. The potentially lower reliability of the negative event narrative markers due to their lower word count might have to some extent constrained their regression weights. Yet, LIWC variables are based on proportions and 50 or less words have proven sufficient to yield reliable estimates in prior research . AlthougDespite these limitations, our findings have important clinical implications. We identified early linguistic markers of later PTSD assessed in narratives provided by survivors at 18 days post-trauma and found that they predicted 7% of later variance in PTSD over and above initial symptom severity. Self-immersed processing assessed by a linguistic index and mental defeat indexed by self-report questionnaire emerged as the best set of early predictors of later PTSD in the present study. Linguistic markers can thus make a clinically significant contribution and help identify those individuals at risk of developing chronic PTSD. Early provision of trauma-focused psychotherapy for these individuals at risk can prevent chronic PTSD . LinguisThe protocol was approved by the IRB of King's College Hospital. All subjects gave written informed consent in accordance with the Declaration of Helsinki.BK and AE developed the study concept and the study design. Testing and data collection were performed by BK. BK, AH, and AE performed the data analysis and interpretation. BK, RK, AH, and MM drafted the paper, and AE and MM provided critical revisions. All authors approved the final version of the paper for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Small non-coding RNAs (sncRNAs) play important roles in health and disease. Next Generation Sequencing (NGS) technologies are considered as the most powerful and versatile methodologies to explore small RNA (sRNA) transcriptomes in diverse experimental and clinical studies. Small RNA-Seq (sRNA-Seq) data analysis proved to be challenging due to non-unique genomic origin, short length, and abundant post-transcriptional modifications of sRNA species. Here, we present Manatee, an algorithm for the quantification of sRNA classes and the detection of novel expressed non-coding loci. Manatee combines prior annotation of sRNAs with reliable alignment density information and extensive rescue of usually neglected multimapped reads to provide accurate transcriptome-wide sRNA expression quantification. Comparison of Manatee against state-of-the-art implementations using real and simulated data demonstrates its high accuracy across diverse sRNA classes. Manatee also goes beyond common pipelines by identifying and quantifying expression from unannotated loci and microRNA isoforms (isomiRs). It is user-friendly, can be easily incorporated in pipelines, and provides a simplified output suitable for direct usage in downstream analyses and functional studies. Other sRNAs identified in NGS experiments, such as ribosomal RNAs (rRNAs), transfer RNAs (tRNAs) and small nucleolar RNAs (snoRNAs), were usually conceived as findings of secondary significance. However, recent studies have provided insight into novel biological roles of such sRNAs7. Using relevant approaches, new sRNA families with biological functions that are still under debate have been discovered. tRNA-derived RNA fragments (tRFs), a novel class of sRNAs second in abundance only to miRNAs5 or box C/D snoRNAs7 comprise characteristic examples of such classes. The majority of tRF sequences are derived from precise cleavage and processing at the 5′ or 3′ end of mature or precursor tRNAs, and studies indicate their possible involvement in miRNA-like RNA targeting as well as global translational suppression8. snoRNAs, known to serve functions in RNA modification processes9, have been recently shown to host specific miRNA-like short RNAs and have been found deregulated in various diseases and malignancies7. Hence, accurate quantification and analysis of the full sRNA spectrum is of great interest.The discovery of short functional RNA classes such as microRNAs (miRNAs) and small interfering RNAs (siRNAs) revealed their involvement in pervasive regulation of gene expression and inaugurated the RNA revolution. NGS techniques offer a powerful high-throughput means for the quantification and discovery of many sRNA classes10. The non-coding RNA (ncRNA) field is rapidly expanding with an increasing number of newly identified and biologically relevant and important ncRNAs11. These considerations highlight the need for sensitive, accurate and efficient bioinformatics tools that can properly handle any kind of small ncRNA present in sRNA-seq datasets.Small RNA-seq data contain a plethora of processing and maturation products potentially including yet unknown RNA species12. As a consequence, the most common approaches adopted for RNA-Seq data13 cannot be successfully applied here: retaining only uniquely aligned mappings14 leads to the omission of a significant portion of reads, while other strategies such as equal distribution16, random read placement17 or reporting all possible alignment positions of multimapping reads18, inevitably leads to incorrect or indirectly quantifiable results13. Additionally, the analysis of numerous intermediate and terminal products of sRNA biogenesis, as well the potential discovery of yet unknown RNA species in sRNA-Seq data, remains undermined with current approaches11.Currently the analysis of sRNA-Seq data is not as mature as for longer RNAs, and their usefulness is impacted by major hindrances. Particularly, the short length  of sRNA-Seq reads introduces the problem of multi-mapping, where a single read may align to multiple genomic locations with equal alignment scores. This issue is exacerbated if we consider that many sRNAs are transcribed from repeat loci13. However, these methods are bound to quantifying only known sRNAs, while reads that could align better in other genomic loci are forced to map with lower scores in the reduced search space19. The ambiguity of the genomic origin of sRNAs may also lead to cross-mappings, in which a short RNA originating from one locus is partially or completely assigned to a different location20. Moreover, most available algorithms are dedicated to studying a single sRNA biotype21, which further restricts the alignment space and can lead to the misclassification of reads.State-of-the-art methods employ direct alignment against known miRNA or sRNA annotations and not on the genome, in order to diminish the extent of multi-mapping21, and those pursuing to cover the broad sRNA space such as miRge22, sRNAbench23, and ShortStack24. miRDeep2 is an extensively used tool dedicated entirely to miRNA quantification, while miRge prioritizes the miRNA biotype over the rest of the sRNAs by utilizing a step-wise alignment strategy against mature miRNAs, miRNA hairpins, ncRNAs, mRNAs, and a modified miRNA library. Implementations such as sRNAbench or ShortStack have sought to address the positioning of multimapping reads in a more refined manner. sRNAbench either assigns multimaps wholly to all their mapping positions, or divides their counts equally between them. Both practices could potentially lead to misinterpretation of transcript expression, especially in cases where multimapping positions pertain to different RNA biotypes. Read mapping, as implemented in the ShortStack tool, is based on local-weighting read alignments. The attempt for improved multimaps placement in ShortStack, relies on unique or fractional weighting schemes and their calculated probabilities for each alignment. This probabilistic placement of multimaps may lead to disparate expression profiles in repeated executions of the same sample. Additionally, highly multi-mapped reads, which can still carry biologically important information, are discarded by this approach.Current implementations for sRNA-Seq quantification can be divided in two categories based on their analysis scope: those that quantify only a single sRNA family, such as miRDeep2Since multimaps are a major obstacle for accurate analysis of sRNA-Seq datasets, we first analyzed 30 sRNA-Seq libraries from diverse tissues to assess the distribution of uniquely aligned and multimapping reads across samples. We further examined the mappings with respect to existing annotation and identified interesting aspects of sRNA-Seq data and assessed the underlying complexity in the placement of multimapping reads. Based on our findings, we implemented the sMAll rNa dATa analysis pipElinE (MANATEE) for detection and quantification of known and unknown small RNAs by efficiently rescuing and utilizing multimapping reads. Manatee is not limited to a single sRNA class and achieves highly accurate results, even for elements residing in heavily repeated loci, by making balanced use of existing sRNA annotation and observed read density information during multi-mapping read placement. Manatee does not prioritize any particular sRNA type, enabling the accurate quantification of diverse RNA classes. Additionally, Manatee exploits sRNA-Seq reads to detect expressed unannotated genomic loci that could harbor still unknown sRNA products. The user-friendly pipeline of Manatee returns ncRNA expression counts that can be directly utilized in downstream analyses, such as differential expression analyses, rendering it easily integrable in larger bioinformatics workflows.25 (GEO). Figure unannotated regions of UAR clusters annotated regions that also contain UAR clusters (green)unannotated regions that also lack UAR clusters (orange)annotated regions and regions with UAR clusters with no concordance (pink).In order to study the characteristics of multi-mapping reads, we performed an initial analysis of 30 distinct human sRNA-Seq libraries derived from hepatoblastoma, liver, brain, gallbladder, colon, lung, pancreas, skin, tongue, thyroid, and heart tissue, embryonic stem cells, as well as MCF7 and HepG2 cell lines, in order to assess the extent of multimaps and uniquely aligned reads (UARs) in sRNA-Seq datasets  in the analyzed datasets mapped to multiple genomic loci Fig. . 19.7% oThe conclusions yielded by the multimaps analysis constituted the basis for the Manatee algorithm which attempts to approach the multimap issue by simultaneously incorporating information from UARs and existing annotation. We aimed to combine into a single step the crucial information of uniquely mapped reads and annotation without prioritizing any particular sRNA type . Bowtie v117 was used as a baseline, since it is a commonly used aligner in sRNA-Seq pipelines, while miRge, ShortStack, and sRNAbench were employed as state-of-the-art approaches in the evaluation. miRge and ShortStack perform read alignment with singular functionality, against sRNA annotation (miRge) and against the genome (ShortStack), while sRNAbench extends the functionality of miRanalyzer by applying genomic/transcriptomic alignment of multiple sRNA types in a hierarchical step-wise manner. Those diverse approaches of sRNA quantification constitute attractive candidates for direct comparisons with the Manatee algorithm. miRge, ShortStack, sRNAbench , and Manatee were executed under their default settings. Bowtie was executed permitting a maximum of 1 mismatch and up to 5 multimaps, while transcript quantification was performed with HTSeq-Count14 using the intersection-nonempty mode and “nonunique all” parameter. The selected parameters for both Bowtie and HTSeq-Count were found to be optimal for the input in question.The accuracy of Manatee was initially evaluated using a simulated short read dataset  Fig. . All tooManatee is not only able to map and accurately quantify diverse sRNA classes, but it also fares favorably when compared to methods specifically designed for miRNAs. miRDeep2, which uses Bowtie to map sequencing reads against precursors and discards or assigns multimaps equally to their valid loci, was executed against the same dataset, with default settings. These results vividly depict that Manatee users can quantify and investigate underexplored small RNA classes, while also obtaining accurate and robust results for miRNAs (the superstar of sncRNA class).The sum of simulated transcript counts was contrasted against the estimated counts by the six tools. ShortStack displayed tendency for count inflation, while HTSeq-Count, miRge, miRDeep2, and sRNAbench underestimated transcript counts Fig. . PrecisiAlthough using simulated datasets offers the advantage of knowing the true transcript abundance, this practice can be prone to shortcomings . The alternative of using real data, allows the examination of agreement among different quantification algorithms. For this reason, we used sRNA-Seq data derived from breast cancer MCF7 cells  and obtained from GEO to cross-correlate the compared sRNA/miRNA quantification methods using Pearson correlation  was observed for the performance of Bowtie + HTSeq-Count and ShortStack, followed by the Manatee-sRNAbench pair-wise comparison (r = 0.77). For miRNAs, Manatee exhibited >0.8 correlation coefficient with ShortStack, sRNAbench and miRDeep2, and exhibited the highest correlation (r = 0.94) with miRDeep2 which is the reference tool in miRNA quantification. When comparing the total sRNA transcriptome results, a substantial divergence between estimated counts was observed across executions. These findings indicate that the tools may each have intrinsic properties that result, at least in some cases, in misclassification and erroneous quantification of sRNAs.µ = 35.06 and σ2 = 114.05). Users aiming to proceed with the detection of novel sRNA genomic loci are strongly advised to first overlap the detected clusters with protein-coding exon annotation in order to exclude putative products of mRNA degradation events26. Following this filtering step and using coding annotation derived from Ensembl v8527, 74 clusters remained as highly promising loci for further investigation.Manatee supports the detection of expressed unannotated loci that can be used to identify novel sRNAs and sRNA classes in diverse research settings. Execution of Manatee with default settings on the MCF7 sRNA-Seq sample  detected a total of 588 unannotated clusters. 503 clusters with cluster length <50 nt are shown in Fig. Small RNA-Seq experimental datasets require extra caution during the alignment and quantification processes compared to RNA-Seq libraries due to technical obstacles arising from small read and transcript lengths. Short sequences tend to map to more than one genomic region, thus affecting transcript quantification. Furthermore, numerous small RNAs  can originate from repeat loci. Variations and post-transcriptional modifications introduce an additional layer of complexity to the detection of proper mapping loci. Many available tools for sRNA-Seq analysis focus on specific short RNA types, at the expense of other subclasses and the breadth of the investigation. Alignments solely against transcripts neglect expression patterns which could bear information about their origin. Alternatively, alignments against the genome are hindered by the lack of unique assignment of numerous sequenced reads. The extensive analysis of small RNA alignments in 30 sRNA-Seq samples revealed that a substantial proportion of sRNA-Seq reads are indeed multimapping. Further analysis showed that many of these positions may refer to different RNA classes, supporting the idea that placement of short reads should include diverse RNA biotypes. Manatee is an algorithm for quantification of sRNA-Seq data based on a novel way of multimaps rescue based on a step-wise approach, exploiting (i) available annotation and (ii) reliable/robust density information towards an optimized multi-mapping read placement which as shown here, improves the accuracy of small RNA quantification. Compared against standard and state-of-the-art methods, Manatee seems to outperform all tested methods even those that are specific to a single sRNA class (e.g. miRNA-specific methods). Furthermore, it enables the detection and quantification of putative expressed small RNA loci lacking annotation. Future expansions of the algorithm could include the incorporation of tolerance against common post-transcriptional modifications or indels to further boost the precision of transcript quantification in a broader and more realistic alignment space. Manatee provides an improved approach to quantify transcripts present in sRNA-Seq data by combining reliable information inferred from UARs and transcript annotation, to more accurately guide the placement of multi-mapping reads. It is an efficient and user-friendly tool that can be a significant aid in small RNA studies.et al.28. In brief, dataset quality control was performed using FastQC29. Cutadapt30 was used for adapter and contaminant removal. Reads were mapped against the GRCh38 human reference assembly using Bowtie. UARs and multimaps with up to 50 genomic positions were retained for further analysis. Clusters of UARs were created across the genome for each sample. UARs were considered as reads mapping uniquely to the genome with one allowed mismatch and Bowtie “best strata”17. Genomic position of UAR includes the information about the mapping chromosome, strand, start, and end position. The minimum density of a UAR cluster was set to one read. Non-coding annotation available in Ensembl v8527, GtRNAdb 2.031, and miRBase v2132 was used to construct a reference for genomic features. Specifically, long ncRNA (lincRNA), mitochondrially encoded rRNA (mt-rRNA), mitochondrially encoded tRNA (mt-tRNA), processed transcript, rRNA, small cytoplasmic RNA (scRNA), snoRNA, small nuclear RNA (snRNA), and vault RNA (vtRNA) gene types were derived from Ensembl, tRNAs were derived from GtRNAdb, while miRNA precursor and mature annotation was derived from miRBase. A minimum 1 nucleotide overlap between the genomic position of aligned read and an annotated transcript was required to assign the read to that specific transcript. All transcripts and UARs were extended by 50nt at each end to allow flexibility in the assignment of reads without adding bias.Quality-check and pre-processing of all libraries utilized in multimaps analysis was performed as in Vlachos Manatee requires FASTQ/FASTA sRNA-Seq data files that have been pre-processed for 3′ adapter and barcode removal. Genomic annotation for ncRNAs is required as input in GTF format with the following tags in the attributes field: gene_name, gene_id, and gene_biotype.xi and yi are the start and end placement positions of the multimap i and r is the range in the close proximity of the read (default 50). Function fcov denotes the UAR density at genomic position p and fprox assigns weights to fcov based on the position p within the genomic region . The multimap is split across its valid multi-mapped loci (MML) according to the score calculated using function fsplit. n denotes the relevance of approximate density distribution and is set by default to 10. For multimaps with non-matching annotation and positioning of UAR clusters, annotation is preferred and used to guide the final placement of the reads. If a multimap falls into regions which are annotated completely or partially, all relevant transcripts are noted in the output file in the form of alternative transcripts. In case where at least one annotated miRNA is present among those features, the read is assigned to the transcript which exhibits the highest coverage score (ratio):The full outline of NGS reads abundance estimation adopted by Manatee is provided in Fig. Coverage is the number of overlapping nucleotides between the annotated feature (transcript) and the read length. The ratio heuristic prioritizes the annotation with the highest coverage, while also considering read and transcript lengths.Reads that exceed the multi-mapping threshold and reads that could not be mapped to the genome are additionally aligned against the transcriptome based on the provided annotation. In the latter case, the number of allowed mismatches is gradually augmented (maximum default 3). In both cases, reads that can be assigned to transcripts with existing mapping densities calculated in previous steps are assigned to those transcripts. If no expression estimates exist, up to five transcripts with the highest mapping quality are retained and assigned as alternative transcripts.All reads assigned and quantified as miRNA type are retained and stored in a separate output file. Each detected putative isomiR sequence is stored independently along with its estimated count. Since each read represents the actual sequence of the sRNA molecule, it can be used to identify diverse miRNA modifications, such as post transcriptional modifications, 5′ and 3′ templated additions, or single nucleotide variations. Manatee saves all reads and clusters them per miRNA. These results may serve as the foundation for a downstream isomiR analysis.UARs mapping to loci lacking genomic features are organized into read clusters based on their genomic positions. Manatee identifies clusters as genomic regions which contain at least five reads and no gap longer than 50 nt between consecutive reads with the default parameters, which can be altered by the user. The output of this step is a single file comprising the unannotated genomic loci and their associated read counts.33, DESeq 234, or edgeR35). Apart from information regarding the quantified transcripts  and estimated non-normalized counts (“Count” column), the files provide also other useful metrics, such as reads per million reads mapped on small RNAs (“RPM”) and uniquely mapped reads (“Unique Reads”). If the reads could have been assigned to a different transcript with equal probability, its ID is provided in the “Alternative Transcripts” column.Manatee execution generates three, tab-separated count files  that can be readily incorporated in downstream analysis pipelines, such as counts-based methods for differential expression analyses  was created using random sampling with a Monte Carlo inversion technique. Human annotation was derived from Ensembl v85, GtRNAdb 2.0, and miRBase v21. Three randomly selected sRNA-Seq libraries (Supplementary Table A simulated short read dataset (Supplementary File.


Molecular similarity is a key concept in drug discovery. It is based on the assumption that structurally similar molecules frequently have similar properties. Assessment of similarity between small molecules has been highly effective in the discovery and development of various drugs. Especially, two-dimensional (2D) similarity approaches have been quite popular due to their simplicity, accuracy and efficiency. Recently, the focus has been shifted toward the development of methods involving the representation and comparison of three-dimensional (3D) conformation of small molecules. Among the 3D similarity methods, evaluation of shape similarity is now gaining attention for its application not only in virtual screening but also in molecular target prediction, drug repurposing and scaffold hopping. A wide range of methods have been developed to describe molecular shape and to determine the shape similarity between small molecules. The most widely used methods include atom distance-based methods, surface-based approaches such as spherical harmonics and 3D Zernike descriptors, atom-centered Gaussian overlay based representations. Several of these methods demonstrated excellent virtual screening performance not only retrospectively but also prospectively. In addition to methods assessing the similarity between small molecules, shape similarity approaches have been developed to compare shapes of protein structures and binding pockets. Additionally, shape comparisons between atomic models and 3D density maps allowed the fitting of atomic models into cryo-electron microscopy maps. This review aims to summarize the methodological advances in shape similarity assessment highlighting advantages, disadvantages and their application in drug discovery. Molecular similarity is a key concept in drug discovery and has been routinely used in the discovery and design of new molecules. It is based on the notion that two molecules often share similar physical properties and biological function if they are structurally similar. This similarity principle has been widely utilized in early phases of drug development to discover new molecules. Virtual screening has been used to filter large databases of compounds to a smaller number based on this similarity principle. Molecular similarity has been also employed to optimize the potency and pharmacokinetic properties of lead compounds based on their structure–activity relationships.There are two components of molecular similarity analysis (1) structural representations and (2) quantitative measurements of similarity between two structural representations. Many types of structural representations have been suggested to measure the similarity between two molecules. These include physiochemical properties, topological indices, molecular graphs, pharmacophore features, molecular shapes, molecular fields etc. Further, there are various methods to quantify the similarity between two structural representations, e.g., Tanimoto coefficient, Dice index, cosine coefficient, Euclidean distance, Tversky index etc. Among these, Tanimoto coefficient   where better mean enrichment for USR was observed , falcipain 2, phosphatases of regenerating liver (PRL-3), p53-MDM2 inhibitors and for phenotypic targets such as colon cancer cell lines established the real-world applicability of USR , a GPU implementation of ROCS has been developed that increased the shape comparison speed by about three orders of magnitude over its CPU implementation. FastROCS is capable of processing up to a million conformers per second on a single NVIDIA Tesla K20 GPU (https://docs.eyesopen.com/toolkits/python/fastrocstk/architecture.html). PAPER, an open source GPU implementation of ROCS algorithm, also demonstrated speed acceleration up to two orders of magnitude on an NVIDIA GeForce GTX 280 GPU over its open source CPU implementation on a Intel Xeon E5345 CPU   method  and setting f  = r, where r is a radial function encoding the distance of surface points from a chosen origin. This function can be determined by deriving an expansion of SH basis function given by:Spherical harmonics (SH) based representations which are expansion of SH functions also allow quantitative description of molecular shapes 2 terms. In general, SH are not rotation translation invariant as magnitude of cl, m change based on the rotation of r. Hence, prior alignment is necessary before comparing the shape of molecules. Efforts were also made to make SH rotation translation invariant   Table . Norms oe) Table . Throughe) Table . In HPCCIn several studies, 3D-Zernike descriptors (3DZD)  is the radial function. Zernike moments are calculated using the following equation:where As Zernike moments are not rotationally invariant, so to make them rotation translation invariant, they are expressed as norm Ligand 3D shape similarity comparison using 3DZD is fast and rotation translation invariant. As no alignment step is required for comparison, it can be utilized as a virtual screening tool to filter a database of compounds based on shape similarity with a query molecule.Overall, surface-based shape similarity methods present attractive options for comparing the shapes of small molecules and macromolecules. They were quite successful in estimating the global and local similarities between macromolecules. However, most of these methods are still in infancy as far as small molecule shape comparison is concerned. Several reasons may have contributed to the lack of interest from researchers in accepting these methods as small molecule shape comparison tools. Surface-based methods such as SH and 3DZD are mathematically complex and involve inclusion of many terms to fully capture the shape of a molecule. Moreover, they are slow in comparison to atomic distance-based shape description and comparison methods while their accuracy in retrieving compounds similar in shape to a query does not match Gaussian overlay-based shape similarity methods. Further, while these methods capture very well the global shape of a molecule, the local shape similarity is not represented comprehensively which is very critical in comparing the shapes of small molecules. However, these methods present several new areas of shape comparison such as comparing shape of ligands with that of binding pockets which may be of immense utility for structure-based design.There are many other approaches of shape representation and methods of similarity measurement in addition to these described above. Another way of representing molecular shape is to use molecular descriptors. Several shape-based descriptors have been traditionally used to compare small molecules and develop QSAR models. These descriptors mostly represent shape implicitly with other properties such as size, symmetry and atom distribution. These include Weighted Holistic Invariant Molecular (WHIM) descriptors of shape  method  has implemented this method in its EM navigator utility to provide shape based structural similarity search against protein databases  has helped researchers to overcome resolution barrier and provide structural and mechanistic insights into structures of difficult proteins and large protein assemblies. Most of these improvements came from the advances in sample preparation, electron detector technologies, improved microscope and computational data processing. Computational methods played an important part in particle picking, particle reconstruction, building and fitting of structures into cryo-EM maps. In recent years, several methods were developed to improve building, fitting and refinement of protein structures in cryo-EM maps (Esquivel-Rodríguez and Kihara, 3D shape similarity methods have contributed immensely to the overall acceptance of the computational virtual screening methods in drug discovery. Most shape similarity methods for shape comparison of small molecules and macromolecules took inspiration from the approaches developed to compare the shapes of 3D objects in computational geometry field. Several approaches were developed ranging from extremely fast atom distance-based methods to comparatively slower mathematically complex methods such as SH and 3DZD. Among all the 3D shape comparison methods, atomic distance-based and Gaussian overlay-based methods are the most widely used. These approaches possess several advantages over surface-based methods. Atomic distance-based methods present an extremely fast way of quickly comparing the shapes of small molecules. This has facilitated the screening of very large libraries of millions of compounds within a few seconds. Moreover, screening large libraries increased the probability of finding novel chemical scaffolds. Furthermore, as most of these methods depend on shape rather than the underlying chemical structure, scaffold hopping can be conveniently achieved. Another possible application of these fast shape similarity evaluation methods would be the clustering of large chemical space to generate quality shape diverse HTS screening libraries. Although Gaussian overlay-based methods are slower than atomic-distance based methods, they are fast enough to allow high throughput virtual screening. GPU implementations of these methods is not very difficult as exemplified by the development of several GPU compatible programs such as FastROCS, PAPER, gWEGA etc. resulting in further increase in the processing speeds. Another advantage with Gaussian-based methods is that they allow visualization as they require alignment of molecules prior to shape similarity calculations. Visualization is helpful in understanding the features responsible for biological activity and critical for the optimization of a molecule especially for the molecules with low structural similarity with query compound. However, a suboptimal alignment can lead to errors in volume overlap calculations and thereby affecting similarity scores and visualization. As alignment is the key component of Gaussian overlay methods, efforts should be focused toward improving molecular alignment. Some of these methods employ chemical features to refine global overlays. As alignment is global optimization problem, molecular alignment could also be improved by employing fast local optimization methods. Both atomic distance-based and Gaussian overlay-based shape similarity methods handle ligand flexibility by employing the conformational ensemble. The performance thus indirectly depends upon conformation generation methods. Current state-of-the-art conformation generation methods still struggle to generate near-native conformations of ligands such as peptidomimetics, macrocycles etc. Development of novel conformation generation approaches utilizing knowledge from experimental databases such as CSD and PDB will steer improvement in performance of shape-based virtual screening approaches. Surface based methods such as SH expansion coefficients and 3DZD are suitable for comparing macromolecules and atomic models with electron density maps, however, comparatively less efforts have been made toward utilizing them in small molecule area. One advantage with surface-based methods is that the protein ligand complementarity search is possible by comparing enclosed shapes of binding pockets and ligands. This will be handy in cases where ligand-based virtual screening methods could not be used due to the lack of active compounds. Finally, shape-based similarity could be used in combination with other ligand and structure-based approaches either in hierarchical or parallel manner to improve hit rate especially for difficult targets.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer XL and handling Editor declared their shared affiliation.


ITGB3, whose expression was abundant in both drug resistance and mesenchymal status, as a promising target to overcome chemoresistance. We also confirmed that depletion of ITGB3 sensitized cancer cells to conventional chemotherapeutic drugs by modulating the NF-κB signaling pathway. Considering the poor druggability of ITGB3 and the lack of feasible drugs to directly inhibit this protein, we took an in silico screening for drugs mimicking the transcriptome-level changes caused by knockdown of ITGB3. This approach successfully identified atorvastatin as a novel candidate for drug repurposing, paving an alternative path to drug screening that is applicable to undruggable targets.Even when targets responsible for chemoresistance are identified, drug development is often hampered due to the poor druggability of these proteins. We systematically analyzed therapy-resistance with a large-scale cancer cell transcriptome and drug-response datasets and predicted the candidate drugs based on the gene expression profile. Our results implicated the epithelial–mesenchymal transition as a common mechanism underlying resistance to chemotherapeutic drugs. Notably, we identified The online version of this article (10.1186/s12943-018-0924-8) contains supplementary material, which is available to authorized users. Recent studies in both in vitro cell and in vivo animal models demonstrated that the epithelial-mesenchymal transition (EMT), a major cause of metastasis, is closely associated with chemoresistance . These aTo elucidate the mechanisms underlying chemoresistance, we examined the gene expression profiles of 804 cancer cell lines, as well as their responses to anti-cancer drugs, using data from Cancer Therapeutics Response Portal (CTRP)  , and induced negative feedback regulators such as NFKBIA and TNFAIP3 , we hypothesized that inhibition of NF-κB signaling would be a key process required for cell sensitization  and suppression of BCL2L1 (encoding BCL-xL) by ITGB3 depletion  and searched for drugs with expression signatures similar to those of ITGB3; specifically, we showed that the transcriptional profile of ATV-treated cells was similar to that of ITGB3 knockdown. Consistently, ATV has a radiosensitizing effect on prostate cancer cells [Most targets responsible for acquired chemoresistance in cancers, identified during extensive molecular mechanistic studies, remain undrugged  due to per cells . Althouger cells , our preBy integrating pharmacogenomics and chemical genomic data, we successfully identified both a therapeutic target and a novel chemosensitizing drug to overcome resistance to multiple chemotherapeutic drugs. Our approach can be applied to a wide range of targets beyond those associated with EMT, paving an alternative path to drug discovery even for undruggable targets.Additional file 1:Supplementary Materials, Methods, and Figures . (PDF 5239 kb)


Abrupt kink in the spinal cord or widened cerebrospinal fluid (CSF) space can be caused by a variety of reasons. The differential considerations include arachnoid web, intradural extramedullary epidermoid or arachnoid cyst, abscess or cystic schwannoma. We discuss the features, imaging, differentials, and treatment of ISCH as a rare cause of such kink in the cord. While reading such cases, a radiologist should include the location, segments involved, cord signal abnormality, visible defect, scalpel sign or C–sign, ventral cord kink, nuclear trail sign, the ventral CSF space preservation, or obliteration and the type.Idiopathic spinal cord herniation (ISCH) is displacement of spinal cord through a dural or arachnoidal defect. Most patients present with back pain or myelopathy, paresthesia, and sensory or motor weakness. Imaging findings include anterior displacement of the cord with possible kink, no filling defect on CT myelography, and no restricted diffusion/mass lesion on  There is no associated history of trauma or surgery. Timely recognition is warranted to prevent permanent neurological deficits. Increased awareness of this clinical condition may result in a timely diagnosis and prompt treatment. In this review, we discuss clinical and an imaging characteristic of this rare pathology followed by a comprehensive review of literature.Idiopathic spinal cord herniation (ISCH) is an uncommonly recognized cause of non-compressive myelopathy in which the spinal cord herniates through a small anterior or lateral dural defect.Clinical and imaging data of three patients with ISCH were analyzed.A 67-year-old female presented with complains slowly progressing bilateral leg weakness with associated chronic low back pain. There was no bowel or bladder involvement. On examination, motor and sensory exams were normal. Lab work was unremarkable. Magnetic resonance imaging (MRI) was performed which demonstrated anterior displacement of the spinal cord with an associated kink at T6 level with cerebrospinal fluid (CSF) pulsation artifacts in the posterior subarachnoid space . High-reA 76-year-old female presented with progressive onset difficulty in walking, leg weakness, and gait ataxia. On clinical exam, she had a sensory level T9 and T8-9 myelopathy was suspected. Lab parameters were within normal limits. There was no history of trauma. MRI thoracic spine demonstrated acute ventral kink in the thoracic spinal cord at T2-3 level .A 52-year-old female presented with progressive onset lower extremity weakness and gait abnormalities. Neurological exam was within normal limits. Lab parameters were unremarkable. She denied a history of trauma. MRI thoracic spine demonstrated acute ventral kink in the thoracic spinal cord at T4-5 level .,3 Several mechanisms have been postulated to cause these dural defects. Remote history of trauma or trivial/occult injury may lead to dural tears. Herniated calcified disk may cause thinning, erosion, or rupture of the dura. In addition, it has been postulated that congenital duplication of dura can provide a potential space for the cord to herniate between the two layers of the dura. ISCH exclusively involves the thoracic spinal cord. This has been suggested due to a limited range of mobility of the thoracic spine and ventral spinal cord curve secondary to of physiologic kyphosis.The etiopathogenesis of ISCH remains unclear; however, the focal dural defect has been blamed for the development of this rare clinical condition. with female predominance (M/F ratio of 3:2). Most common clinical presentation is Brown-Séquard syndrome, in greater than half of the reported cases. Other major early manifestations may include numbness and decreased temperature sensation in the legs, gait disturbances, pain, and incontinence.,8ISCH presents in 22–71 years of ageMRI is the imaging modality of choice for diagnosing ISCH. Usually, there is a single dural defect; however, there have been reports of two defects.,10 The herniated part of the cord might appear as a small mass with a signal [,11 Occasionally, abnormal cord signal abnormality may be seen representing syrinx formation [ Scalloping of the posterior vertebral body may also be noticed. The end plate irregularity and/or sclerosis or herniated disc calcification called “Nuclear trail Sign” can be seen sometimes on CT scan.An abrupt anterior kink may be seen, called the “Scalpel Sign” [ This is best appreciated on high-resolution T2-weighted images such as constructive interface in steady state or sampling perfection with application-optimized contrasts using different flip angle evolution.,15 When the findings on MRI are equivocal, a CT myelography should be performed. It shows free migration of contrast through the dural defect at the level of the herniated segment or may show the widened dorsal subarachnoid space.,16-18 The phase contrast pulse cine MRI demonstrates normal pulsatile CSF flow in subarachnoid space posterior to herniated cord in ISCH differentiating it from posteriorly positioned intradural arachnoid cyst.The visualization of nerve roots traversing the dorsal subarachnoid space at the level of the cord deformity helps to differentiate SCH from other pathologies posterior to cord displacing it anteriorly such as a subarachnoid cyst or epidermal cyst which instead displaces the nerve roots peripherally.,19A classification system for ISCH has been reported based on the severity of herniation which helps in pre-operative planning and also in prognosticating patients.Type K: Kink toward the ventral region.Type D: Cord disappears at the herniated site.Type P: Protrusion of the cord in a way that the ventral subarachnoid space is effaced with very little posterior kink.Type C: When the hiatus is central.Type L: Hiatus is lateral.The Type P has good post-operative recovery. Type C hiatus with bone defect have severe pre-operative symptoms and poor post-operative outcomes.,21 Surgery is laminectomy with intradural adhesiolysis and arachnoid band resection with verification of flow of CSF from cranial and dorsal ends of dural opening followed by closure of dural defect with fat or dural patch or widening of the dural defect. Surveillance is recommended for either form of treatment.The treatment of ISCH includes conservative management or surgery depending on the clinical presentation. Surgery is recommended with severe neurological symptoms or progressive worsening with the aim to reduce the herniated cord and prevent its recurrence.ISCH is a rare clinical entity with a displacement of the spinal cord through a dural or arachnoidal defect. Early recognition of this abnormality is necessary to prevent permanent neurological deficits. An important imaging finding is anterior displacement of the cord with a kink, without associated mass lesion.


Mitragyna speciosa), a plant native to Southeast Asia, contains the alkaloid mitragynine, which can produce stimulant effects in low doses and some opioid-like effects at higher doses when consumed  of these decedents tested positive for kratom on postmortem toxicology (kratom-positive). Postmortem toxicology testing protocols were not documented and varied among and within states. Kratom was determined to be a cause of death  by a medical examiner or coroner for 91 (59.9%) of the 152 kratom-positive decedents, including seven for whom kratom was the only substance to test positive on postmortem toxicology, although the presence of additional substances cannot be ruled out (In approximately 80% of kratom-positive and kratom-involved deaths in this analysis, the decedents had a history of substance misuse, and approximately 90% had no evidence that they were currently receiving medically supervised treatment for pain. Postmortem toxicology testing detected multiple substances for almost all decedents . FentanyKratom-positive deaths accounted for <1% of all SUDORS overdose deaths during July 2016–December 2017. Identification of kratom is method-dependent (The type and number of substances detected in kratom-involved deaths can inform overdose prevention strategies (


Pelecanus occidentalis californicus) following oil contamination and rehabilitation to test for differences between previously oiled and rehabilitated (post-spill) and presumably uncontaminated pelicans. Post-spill pelicans released with either color leg bands alone, or bands plus harness-mounted satellite GPS tags, were relocated and visually assessed in the field at non-breeding communal roosts and compared to surrounding unmarked pelicans in the general population. Non-oiled pelicans bearing GPS tags were also included in the study. Post-spill pelicans lagged the general population in molt of ornamental yellow crown feathers but hind neck transition into white plumage was not significantly different. Both post-spill and non-oiled pelicans wearing GPS tags had lower gular redness scores than the unmarked, non-oiled population. Pre-breeding gular pouch redness of post-spill pelicans was more strongly influenced by wearing of a GPS tag than a history of oil contamination and rehabilitation. Gular pouch redness of post-spill pelicans in the first 18 months after release was positively correlated with long term survivorship. If gular pouch color is a condition-dependent sexual signal and overall health influences plumage molt progression, our results indicate that many post-spill pelicans marked with bands alone were in relatively good condition going into the next breeding season, but those released with electronic tags experienced additional stress due to wearing the equipment, introducing a confounding variable to the post-release study.Sublethal effects of oil spills may dampen seabird rehabilitation success due to lingering negative impacts of contamination and stress on reproduction and long-term survival. These effects can be difficult to measure while birds are in care as well as once birds are released. Expression of sexually selected traits that are sensitive to condition can provide information on physiological status of birds. We evaluated plumage molt and gular pouch skin color of California brown pelicans ( Wildlife exposed to petroleum products after spill incidents can be negatively affected in multiple ways. The primary acute impact to these animals is from external contamination by oil and subsequent hypothermia, increased metabolic effort, and emaciation –4. IngesResearchers have used a variety of techniques to track the fate of oiled seabirds following rehabilitation and release. These include quantifying band recoveries of live and dead birds, monitoring movement patterns with electronic tracking, and documenting reproductive success , 14–17. Pelecanus occidentalis) are a seabird commonly oiled following nearshore spills in the United States. Survival and dispersal of previously oiled and rehabilitated California brown pelicans  were examined in the early 1990’s by Anderson et al. [P.o. carolinensis) successfully breeding one year after oiling from the Deepwater Horizon spill. An increasing number of studies have demonstrated that seabird oil spill rehabilitation efforts can succeed in restoring birds to normal function, but further evidence is needed–specifically projects that extend beyond acute survival [Brown pelicans  during the breeding season has been related to a combination of carotenoids, hemoglobin, and increased blood flow [Both sexes of California brown pelicans (hereafter ‘pelicans’) develop a red gular pouch prior to the breeding season, when the dull grayish gular pouch blooms with redness proximally . The pouood flow . The speLarus michahellis) beaks was smaller in birds exposed to polyaromatic hydrocarbons compared to those that were not exposed. Similarly, the hue and brightness of skin colors of kittiwakes during the breeding season showed a negative relationship to endogenous pollution levels [Coloration has been found to indicate health and fitness in many species of birds , 43. Carn levels .We designed a study to test the null hypothesis that post-spill pelicans observed in the wild would not differ visually from conspecifics with respect to their outward physical appearance. Here, we provide a field-based comparison of molt progression in the head and neck, and expression of redness in the gular pouch between previously oiled/rehabilitated pelicans and the general, presumably never oiled, wild population. We further report on unexpected differences between post-spill pelicans bearing electronic tags versus post-spill birds released with only leg bands, and the relationship of gular color to known survival amongst rehabilitated birds.To further improve estimates of seabird recovery following oiling, the Oiled Wildlife Care Network (OWCN), a program led by the School of Veterinary Medicine at the University of California at Davis, initiated a study to track rehabilitated brown pelican movement patterns and survival following the May 2015 Refugio Beach Oil Spill near Santa Barbara California using Global Positioning Satellite platform transmitter terminal (GPS-PTT) tags . We deveThe Refugio Beach Oil Spill occurred on 19 May 2015, in Santa Barbara County, California, USA at 34.46° N, 120.09° W . The spiWork was completed in accordance with all appropriate State, Federal and university regulations and policies including the U.S. Geological Survey Bird Banding Laboratory with Federal Master Bander permit #23539, the U.S. Fish and Wildlife Service Scientific Collection Permit #MB191637-0, and the California Department of Fish and Wildlife Scientific Collection Permit #SC-003855. All procedures were conducted under University of California at Davis Institutional Animal Care and Use Committee Protocol #18823. Brown pelicans are protected under the Migratory Bird Treaty Act.Field reconnaissance was initiated approximately three months after release of the previously oiled birds. Data collection on plumage and color comparisons took place primarily from September 2015—March 2016. Field work occurred in seasonal sessions of 7–10 days and ranged along the coast from Grays Harbor, Washington, USA  to Ojo de Liebre, Baja California Sur, MX  previously oiled pelicans released with color band-only, 2) previously oiled pelicans with band plus GPS-PTT, and 3) non-oiled pelicans with band and GPS-PTT. Plumage aspect and soft-part color characteristics were based on classifications in Schreiber et al. .Molt phase and color assessments were limited to adult birds and focused on three distinct and readily visible body parts—the hind neck, the crown feathers, and the gular pouch. These features were visually scored using a combination of direct field observations and digital photographs. The hind neck and crown were categorized as either in a definitive plumage aspect or transitioning between states . An ordiSurveys of banded pelicans to document survival continued through the summer of 2018 and each post-spill bird was photographed. A separate analysis of post-spill survival of the Refugio incident pelicans, incorporating demographic and biomedical factors, is planned .Statistical analyses were conducted using R package v. 3.5.1(R Core Team 2018) . PelicanMost of the Refugio post-spill pelicans (79%) were re-sighted at least once following release. By September 2017, 220 sightings of 33 birds were documented . Frequent surveys of large communal roosts resulted in a greater number of visual encounters of target animals than did attempts to relocate electronically tagged pelicans based on GPS-PTT data feeds showing past positions. Sightings of the post-spill birds were most common in summer and fall  and relatively rare during late winter-spring  when pelMost visual sightings of post-spill birds occurred over 300 km north of the spill region in Central California . This waPlumage of post-spill band-only pelicans appeared normal with respect to waterproofing and overall integument condition , while four of the 12 pelicans seen wearing transmitters were observed shivering . Plumage disruption around the harness was generally visible around the ventral surface  and exceDuring the mid-September—December sampling period, nearly all non-oiled pelicans (98%) displayed solid yellow crown plumage . EmergenMost pelicans (93%) in the non-oiled general population had definitive basic white neck plumage during the sampling period , althougPelicans began to display red pouches at nonbreeding roosts by mid-September 2015, and red pouches were observed through March 2016. Expression of redness in the non-oiled/unmarked population was variable each month within the sampling period but scores tended to be highest in November-February . PreviouS = 0.796, p < 0.001). None of the birds with gular scores of zero (no redness in the pouch) were encountered >247 days post-release. Gular redness scores of zero were also associated with known mortality. Of four confirmed pelican mortalities in this study, three were seen alive in the field during the period of expected pouch redness, and all had a score of zero. Three of the pelican carcasses were GPS-PTT-bearing and found by searching in areas where electronic signals suggested lack of normal movement . The fourth carcass was from a color band-only bird and found incidentally by a member of the public.Sightings of banded birds through summer 2018 indicated high survival rates of the Refugio post-spill pelicans overall; at least 60% (25 of 42 pelicans) were alive one year after release and 43% (18 pelicans) were seen alive three years after the spill. Long-term survival of adult post-spill birds was positively correlated with the highest GRI score documented in the first 18 months after oil exposure  fitted with dummy transmitters attached with Teflon ribbon harness had lower body mass than controls without equipment. Fitted mallards also tended to avoid water, presumably due to greater energetic expenditure to thermoregulate in cold weather and compromised feather insulation caused by the equipment. In our study, one of the equipment-related stressors for pelicans was likely similar bodily heat loss due to plumage disruption around the chest harness and transmitter  apparently incurs oxidative costs for both sexes [If one assumes that the red gular pouch provides a positive measure of health in the California brown pelican, our results suggest that, although not equal to the general population, the post-spill birds released with only bands were in relatively good condition going into the first breeding season after the spill, that numerous complex interacting physiological mechanisms were functioning, and that many of these birds could afford the additional costs of color display , 69. Devth sexes . Perhapsth sexes –72. The th sexes  found thRelationships between body condition, integument color, and breeding success are relatively well-known for several seabird species but are not fully understood in the brown pelican. Investigation of the underlying mechanisms surrounding gular pouch color expression and how it relates to pelican fitness would increase the utility of using pouch color in future studies. In the sexually dimorphic great frigatebird, for example, the male displays a bright red inflated throat pouch during courtship that rapidly fades to orange at the onset of incubation. Astaxanthin, a carotenoid pigment, has been found in very high concentrations in the outer dermal layer of the pouch . The speTargeted field surveys with large sample sizes and from wide geographic ranges are also needed to better establish or update baseline patterns and variation in molt and seasonal color change for California brown pelicans. Schreiber et al.  noted thChronology and outcome of annual breeding effort can affect timing of avian molt , 38 but Despite the challenges, field tracking and visual assessment of plumage and gular pouch color of marked brown pelicans has good potential for future ecotoxicology and post-release studies. The expression of gular pouch redness likely plays a role in behavioral ecology at non-breeding communal roosts, as well as breeding colonies, and may provide a valuable window into many aspects of California brown pelican health and fitness. This study also serves as a reminder to use caution when interpreting data based on animals bearing a significant equipment burden and will hopefully help encourage development of improved remote tracking techniques for brown pelicans.S1 Fig(PPTX)Click here for additional data file.S2 Fig(PPTX)Click here for additional data file.S1 Datafile(CSV)Click here for additional data file.


Plasmodium infections remains a global public health concern. Only a fraction of infected people develops severe clinical syndromes motivating a longstanding search for genetic determinants of malaria severity. Strong genetic effects have been repeatedly ascribed to mutations and allelic variants of proteins expressed in red blood cells but the role of inflammatory response genes in disease pathogenesis has been difficult to discern. We revisited genetic evidence provided by inflammatory response genes that have been repeatedly associated to malaria, namely TNF, NOS2, IFNAR1, HMOX1, TLRs, CD36, and CD40LG. This highlighted specific genetic variants having opposing roles in the development of distinct malaria clinical outcomes and unveiled diverse levels of genetic heterogeneity that shaped the complex association landscape of inflammatory response genes with malaria. However, scrutinizing genetic effects of individual variants corroborates a pathogenesis model where pro-inflammatory genetic variants acting in early infection stages contribute to resolve infection but at later stages confer increased vulnerability to severe organ dysfunction driven by tissue inflammation. Human genetics studies are an invaluable tool to find genes and molecular pathways involved in the inflammatory response to malaria but their precise roles in disease pathogenesis are still unexploited. Genome editing in malaria experimental models and novel genotyping-by-sequencing techniques are promising approaches to delineate the relevance of inflammatory response gene variants in the natural history of infection thereby will offer new rational angles on adjuvant therapeutics for prevention and clinical management of severe malaria.Despite significant progress in combating malaria in recent years the burden of severe disease and death due to  Plasmodium protozoan parasites that develop in multiple stages within the vertebrate host. The chronology of infection exposes the host to distinctive intracellular and extracellular forms that emerge sequentially during the natural history of infection  and by variations in the parasite antigenic complexity represented by stage-specific sets of antigens or by mechanisms of antigenic diversification and clonal antigenic variation –9. As a The host response to malaria includes a wide range of both cell-intrinsic and systemic mechanisms but the initial responses in a naive host are mostly driven by non-specific reactions . Liver sAn archetypal infection in a naive host leads to exponential increase blood stage parasites due to the cyclic expansion of asexual parasite forms  but in nPlasmodium antigens expressed in the surface of infected erythrocytes  through innate immunity receptor signaling  leading to release of pro-inflammatory mediators, namely TNF-alpha, IL-1β, IL-6, and IFN-gamma –50. In oPlasmodium infections develop severe clinical forms. Compelling genetic evidence collected in recent years shows that malaria contributed to shape the human genome and it is established that variants in genes coding for proteins expressed in erythrocytes are major genetic determinants of resistance to infection and resilience to severe disease  is a potent pro-inflammatory mediator involved in various steps of immune responses. It has long been recognized that TNF takes part in anti-eduction . Yet, TNeduction –78. Eleveduction  and was eduction . The TNFeduction , 82. Neveduction  implyingeduction .P. falciparum IgG levels  that control gene expression or TNF production –92 are aG levels . This suG levels –102 and G levels , 104  is a free radical involved in multiple biological processes, including inflammatory responses. Decreased NO bioavailability in severe malaria patients is a well-established pathogenesis factor , 110 posThe NOS2 gene codes for the inducible NO synthase (iNOS) and has been probed in several genetic studies that collectively uncovered common genetic variants functionally correlated with increased NO bioavailability in infected individuals –119. MeaThe protective effects of NOS2 variants have not been consistently observed in different studies possibly owing to differences in the genetic variants analyzed, in the ethnic groups studied or in regional epidemiologic patterns of clinical malaria , 127. Nein vitro and in mice converge at the notion that sensing malaria parasites via innate immune receptors is linked to a IFN-1 response  or in components of the IFN-I induction pathways  (Type 1 interferons (IFN-1) are secreted by immune and non-immune cells including lymphocytes, macrophages, dendritic cells, fibroblasts, and endothelial cells. IFN-1 production is induced upon innate recognition of exogenous nucleic acids and proteins by innate receptors such as membrane-bound Toll-like receptors (TLRs) and cytosolic RNA helicases. Studies response –130. A mnd Irf3) –131. Mound Irf3) , 24; (iind Irf3) –135; andnd Irf3) , 137.Together these findings raise the possibility that IFN-1/IFNAR1 signaling exerts beneficial effects in early stages of infection when innate immune responses dominate. In contrast, detrimental effects are expected at later stages if IFN-1 signaling intrudes in the mounting of adaptive immune responses by impairing anti-parasite responses driven by CD4T cells or exacerbating cytotoxic CD8T cell-mediated reactions leading to tissue damage.IFNAR1) gene polymorphisms associated with mild malaria, disease severity and cerebral malaria in children  in otherwise resilient mice and was abrogated by HO-1 induction or carbon monoxide administration  is a catabolic enzyme that cleaves heme to generate biliverdin, carbon monoxide, and ferrous iron . HO-1 acstration . Tissue stration . In sharnfection .HMOX1 gene showed that polymorphisms functionally controlling HO-1  expression or activity  or hemozoin-DNA complexes can be mediated by several TLRs, providing sensor redundancy in the immuno-detection of blood stage parasites  are ancient components of the immune system that recognize pathogen associated molecular patterns (PAMPs) and are infection sensors at the onset of innate immune responses. Conceivably, TLR signaling could be involved both in resolving early infection and in heightening inflammation and immunopathology. It has been shown that binding to IE and recognition of arasites , 49, 153arasites , 154–160arasites . Togetheoduction . Downstroduction , 161. Fu malaria . The assP. falciparum-infected red blood cells balances between parasite expansion and host responses that in turn drive different clinical outcomes. The search for host genetic factors governing the development of clinical malaria phenotypes has attracted significant research efforts in the recent years. Under the selective pressure imposed by severe malaria, genetic variants with strong effects surfaced in endemic regions and were revealed in genes coding for proteins expressed in erythrocytes, prominently hemoglobin .In contrast, inflammatory response genes associated with malaria show smaller genetic effects but unveiled key steps of the host response in the course of infection namely, innate recognition, phagocytosis, exacerbated inflammation, endothelial activation, and immune adaptive responses . Thus, iThe scrutiny of inflammatory genes genetic variants associated with malaria reveals a complex genetic architecture with multiple levels of genetic heterogeneity, namely:Alternative alleles in one gene either favor or counteract severe disease development , offering direct interpretation of their genetic effects;Different genetic variants within a gene promoter are associated with different severe malaria syndromes  suggesting differential gene regulation in context of different inflammatory milieus;Alleles conferring both resilience to severe disease and susceptibility to asymptomatic infection/mild malaria  suggesting that advantages of selecting for anti-inflammatory alleles carry fitness costs in infection susceptibility and chronic infection;Regulatory variants acting prior infection or during infection map in different gene regions , suggesting that infection status alters gene regulatory regimens.Genetic variants that show disparate roles in malaria pathogenesis depending on their homo/heterozygous state .In this scenario, evaluation of genetic effects requires that the overall allelic variation within an entire gene is taken into account. New genotyping by sequencing methodologies that generate long reads in single DNA molecules enables access to real-haplotype data across entire genes. Data on entire gene variants will allow a comprehensive analysis of their genetic effects in context of genotype combinations with other genes. These data will open new approaches to study non-allelic genetic interactions  and to dissect the genetic architecture of inflammatory responses to malaria.In conclusion, detailed analysis of genetic effects of inflammatory response gene variants is a key step in malaria research to motivate experimental investigations of the underlying pathogenesis mechanisms. This knowledge will be critical to identify rational adjuvant therapies to prevent fatality or undesired malaria complications and subsequent long-term sequels that represent a high burden in endemic regions.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Solitary plasmacytoma of the skull, a single malignant monoclonal plasma cell proliferation without systemic involvement, is rare and often misdiagnosed by radiological examinations only. In this article, the authors presented a 40-year-old man who had a painless protruding mass over the midline of the posterior head region. A brain magnetic resonance imaging (MRI) revealed an enhanced mass lesion over the midline of the parieto-occipital region with skull erosion. Under the tentative diagnosis of meningioma, craniectomy was performed with en bloc tumor resection, and the skull defect was replaced by cranioplasty with bone cement. The final histopathological report revealed plasmacytoma without evidence of multiple myeloma. No further adjuvant radiotherapy was arranged for the patient. The postoperative course was uneventful within a one-year follow-up period. For the skull solitary plasmacytoma, there was no strong evidence that adjuvant radiotherapy was necessary after the primary surgery. Surgical intervention with total tumor resection is an effective option for the patient with solitary plasmacytoma of the skull. Solitary bone plasmacytoma, a single lesion with malignant proliferation of monoclonal plasma cells in the bone, is rare and affects less than 5% of patients with plasma cell myeloma -3. The pA 40-year-old man had noticed a painless protruding mass under the midline parietal-occipital scalp for approximately one year. On plain radiograph Figure , there wHypervascularity of the lesion was suspected based on the MRI with strong enhancement. Angiography revealed dense stains with distorted vessels over the same location Figure . EmbolizHistopathology demonstrated plasmacytic type plasmacytoma with positive lambda stain Figures , 2B. SysIn this study, we presented a 40-year-old man with a solitary plasmacytoma at the midline parietal-occipital region of the skull. The patient was treated by en bloc tumor resection and cranioplasty by bone cement. Radiotherapy was not performed because there was no residual tumor, and chemotherapy was also not arranged since there was no systemic involvement.Solitary plasmacytoma of the skull is rare. The image features of solitary plasmacytoma of the skull included osteolytic lesion with well-defined margin, non-sclerotic borders in a computed tomography (CT) scan, and typically T1 hypointense and T2 hyperintense in MRI with enhancement after contrast injection . BecauseUnlike multiple myeloma, there is no systemic involvement in patients with solitary bone plasmacytoma. There were several recommended diagnostic criteria for solitary bone plasmacytoma, including single area of bone destruction due to clonal plasma cells; normal marrow without clonal disease; normal results on a skeletal survey and magnetic resonance imaging of the spine, pelvis, proximal femora, and humeri; no anemia, hypercalcemia, or renal impairment attributable to myeloma; and absent or low serum or urinary level of monoclonal protein and preserved levels of uninvolved immunoglobulins .The related literature reviewed for the patients who had solitary plasmacytoma and received gross total resection including craniectomy and cranioplasty are listed in Table Definitive local radiotherapy is the treatment of choice for solitary bone plasmacytoma . TreatmeHowever, solitary plasmacytoma may present as an early manifestation of multiple myeloma in some cases. Therefore, frequent measurements of myeloma protein for at least six months after treatment are required to confirm disease radiosensitivity , 2.Solitary plasmacytoma is a rare solitary skull lesion with malignant monoclonal plasma cell proliferation. Gross total resection alone may be an effective treatment option, but solitary plasmacytoma of the skull requires continuous follow-up.


The self-renewal and differentiation potentials of stem cells are dependent on amino acid (AA) metabolism. We review the literature on the metabolic preference of both cancer and noncancer stem cells. The balance in AA metabolism is responsible for maintaining the functionality of noncancer stem cells, and altering the levels of AAs can influence the malignant biological behavior of cancer stem cells. AAs are considered nutrients participating in metabolism and playing a critical role in maintaining the activity of normal stem cells and the effect of therapy of cancer stem cells. Targeting AA metabolism helps inhibit the stemness of cancer stem cells and remodels the function of normal stem cells. This review summarizes the metabolic characteristics and regulation pathways of AA in different stem cells, not only from the nutritional perspective but also from the genomic perspective that have been reported in the recent five years. In addition, we briefly survey new therapeutic modalities that may help eradicate cancer stem cells by exploiting nutrient deprivation. Understanding AA uptake characteristics helps researchers define the preference for AA in different stem cells and enables clinicians make timely interventions to specifically target the cell behavior. Stem cells are poorly differentiated cells with self-renewal ability and can be divided into cancer stem cells (CSCs) and normal stem cells based on their cell proliferation ability and into pluripotent, multipotent, and monopotent stem cells based on their differentiation potential. Pluripotent stem cells, such as embryonic stem cells (ESCs), differentiate into various types of tissue cells, and the stability of this differentiation process maintains the normal growth and development of the human body. CSCs have unlimited proliferation capacity and are closely related to the recurrence, metastasis, and drug resistance in tumors; few CSCs induce tumor occurrence , 2.α (HIF-1α) and return to a stem-like phenotype through dedifferentiation [α, WNT, and Snail, regulate cellular metabolism, and the EMT-related metabolites glutamine, glutamate, and alanine and high lactate concentration are associated with poor survival and high metastatic potential in patients with breast cancer [Because they have high heterogeneity, eliminating CSCs may represent a permanent cure for cancer –5. Tumorntiation , 15. Whentiation . Hypoxiantiation . EMT-relt cancer , 19. Thet cancer . TherefoWe focus on the importance of amino acid (AA) metabolism to the properties of stem cells. This review elaborates the function of AA metabolism in the CSCs and normal stem cells. Defining the metabolic characteristics of different types of cells may increase the specificity of cancer treatments.β-catenin, NOTCH, NF-κB, and JAK/STAT, facilitate such phenotypic plasticity. Some cytokines, such as the vascular endothelial growth factor and epidermal growth factor, are positively correlated with the reprogramming of stem cell properties [The status of cancer cells can be reprogrammed through metabolic remodeling while their dedifferentiation ability for the induced stem-like phenotype is maintained –22. Thisoperties –27. The operties , 24, 28.operties . In addioperties –21. Extrβ-catenin signaling pathway [β-catenin signaling pathway. The above content  is a metabolic enzyme used in serine synthesis, and overexpression of PHGDH has been associated with mortality of patients with breast cancer . In receGlutamine promoted CSCs' proliferation and maintained stemness phenotype. Glutaminase 1 (GLS1) was highly expressed in CSCs and promoted the expression of acetaldehyde dehydrogenase (ALDH); its overexpression induced glutamine metabolism. BCAT1 promoted HSC proliferation and survival via maintenance of amino acid balance. SLC7A11, which function as cystine/glutamate antiporter, was also highly expressed and enhances the stemness phenotype of glioma stem cells (GSCs). Phosphoglycerate dehydrogenase (PHGDH) promoted serine synthesis and maintained cell proliferation and metastasis ability via the maintenance of redox homeostasis. Tryptophan depletion preserved the CSC phenotype via inducing the enhancement of OCT4 transcription.Metabolic drugs are used to treat tumors on the basis of differences in the expression of substances in tumor cells and normal tissues; they mainly target a specific AA or a key metabolic enzyme for dietary or drug intervention. With increasing numbers of studies investigating the effects of AA metabolism on the regulation of tumor stem cell phenotypes, targeting AA metabolism pathways will become a popular topic in cancer therapy research. Although AA metabolism pathways have a strong impact on the properties of CSCs, limited studies have evaluated the treatment of tumors by targeting AA metabolism pathways. Here, we summarize some potential therapeutic modalities .Targeting the function of membrane transporters in CSCs is a potential new therapeutic strategy. In vivo and in vitro experiments have demonstrated that pharmacological deletion of the cystine/glutamate transporter xCT causes AA starvation in estrogen receptor-positive breast cancer cells and suppresses the proliferation of these cells . ActinomActinomycin D (ActD), sulfasalazine target xCT, and CB-839 target GLS to reduce glutamate synthesis. NCT-503 targets PHGDH to reduce serine synthesis. MRK003 targets Notch to induce the threonine level.ESCs are typical pluripotent stem cells with the potential to form complete individuals. Studying the metabolic characteristics of ESCs has been critical to the development of regenerative medicine. Due to ethical constraints, few studies have investigated human ESCs (hESCs); instead, mouse ESCs (mESCs) or induced pluripotent stem cells (iPSCs) have been employed. iPSCs are of a similar type to ESCs. In 2006, Takahashi and Yamanaka  used virIn recent years, researchers have discovered that some AAs are highly sensitive to maintaining stem cell pluripotency and differentiation ability. Therefore, the AA metabolism characteristics of stem cells may be used to improve the induction rate of iPSCs and application security. Since 1998, researchers have found that the expression of stem cell marker  is reduced when cells are cultured in Dulbecco's modified Eagle medium that lacks threonine. Subsequently, the impact of AA metabolism regulation on embryonic development was discovered . When thKilberg et al.  compreheThreonine promoted cell survival via metabolism by TDH to glycine, but excessive glycine inhibited cell differentiation by blocking proline. Threonine input maintained the properties of stem cells including stemness phenotype and pluripotency via upregulating H3K4me2 and c-Myc and Oct4 expression. Threonine induced cell proliferation by activating signal pathways . Adding proline to the medium promoted the differentiation of mouse embryonic stem cells into mesenchymal stem cells. Double depletion of arginine and proline reduced the expression of Nanog and Oct4 in human embryonic stem cells. Methionine deprivation reduced Nanog expression via activating the P53 signal pathway.Hematopoietic stem cells (HSCs) are the original cells in blood, have the potential to differentiate into various blood cells, are regarded as typical multipotent stem cells, and play a key role in maintaining the stability of blood components. At present, HSCs are mainly used to treat leukemia through bone marrow transplantation. However, such transplantation causes difficulties such as immune rejection and metabolic disorders.An imbalance of AA metabolism can affect the proliferation and survival of HSCs in vivo and be harmful to them. Next, Wilkinson et al.  employedHSCs highly expressed glutamine transporter (ASCT2). ASCT2 activated glutamine metabolism resulting in cell differentiation into erythrocyte. Blocking glutamine metabolism resulted in cell differentiation into monocytes. SHP-1 reduced phosphorylation of tyrosine and induced hematopoietic reconstitution ability. Inputting N-acetyl-L-cysteine promoted bone marrow transplantation via inhibiting oxidative stress.α to maintain the stem-phenotype of CSCs, and the expansion of myeloid progenitors induces hypoxia due to oxygen depletion and stabilizes HIF-1α in the bone marrow microenvironment; thus, hypoxia-induced HIF-1α activation is essential to HSC mobilization [After performing synthetic analysis, we discovered that threonine, proline, and methionine affect the process through which ESCs and iPSCs differentiate into different tissues; however, HSCs and CSCs are highly sensitive to glutamine. This may be due to the preference of HSCs and CSCs to an acidic niche. An acidic environment enhances drug resistance by reducing chemotherapeutic drug permeability and promoting drug efflux . In addilization , 87. Thelization . Tryptoplization . HoweverUnderstanding the metabolic expression profiles of different tissues and organs can help researchers to achieve the objective of differentiation from stem cells into specific tissue types according to different preferences of tissues and organs for various AAs; this can be achieved by altering the nutritional input or gene regulation. Stem cells derived from different pathological types have different AA metabolism patterns, which may be related to their microenvironment and genetic background. Therefore, according to the metabolic characteristics of different CSCs, more antitumor modalities against specific CSCs can be developed. The metabolic differences between stem cell types provide a theoretical basis for developing effective antitumor drugs that do not damage normal cells . Further


Platelet-rich fibrin (PRF) therapy has been widely applied in regenerative dentistry, and PRF preparation has been optimized to efficiently form fibrin clots using plain glass tubes. Currently, a shortage of commercially available glass tubes has forced PRF users to utilize silica-coated plastic tubes. However, most plastic tubes are approved by regulatory authorities only for diagnostic use and remain to be approved for PRF therapy. To clarify this issue, we quantified silica microparticles incorporated into the PRF matrix. Blood samples were collected into three different brands of silica-containing plastic tubes and were immediately centrifuged following the protocol for advanced-PRF (A-PRF). Advanced-PRF-like matrices were examined using a scanning electron microscope (SEM), and silica microparticles were quantified using a spectrophotometer. Each brand used silica microparticles of specific size and appearance. Regardless of tube brands and individual donors, significant, but not accidental, levels of silica microparticles were found to be incorporated into the A-PRF-like matrix, which will be consequently incorporated into the implantation sites. Presently, from the increasing data for cytotoxicity of amorphous silica, we cannot exclude the possibility that such A-PRF-like matrices negatively influence tissue regeneration through induction of inflammation. Further investigation should be performed to clarify such potential risks. Similar to platelet-rich plasma (PRP), platelet-rich fibrin (PRF) has been demonstrated to be effective in tissue repair/regeneration in various fields of regenerative medicine ,2,3,4,5.However, PRF users have recently faced a serious problem where, except for some brands specialized for PRF preparation, major medical equipment manufacturers have discontinued the production of plain glass tubes for blood collection . TherefoUpon contact with the glass surface, coagulation factor XII in the plasma can be activated to consequently produce a fibrin clot through the activation of the intrinsic coagulation cascade . Since tHowever, such hybrid plastic tubes are not yet available commercially, and therefore many PRF users employ existing products as replacements of the original plain glass tubes. Among the existing products, the plastic tubes for blood coagulation meet their requirements . AlthougTo evaluate the quality of the PRF prepared using silica-coated plastic tubes, we characterized the morphology of the silica microparticles and examined their possible contamination in the resulting PRF matrix.After obtaining individual written informed consent, blood samples were collected, without anticoagulants, from six non-smoking healthy male volunteers aged 30–60 years. The study design and consent forms for all procedures (project identification code: 2297) were approved by the ethics committee for human participants at the Niigata University School of Medicine  on 14 October 2015, in accordance with the Helsinki Declaration of 1964, as revised in 2013.® serum clot activator tubes  and Neotube® celite tubes  or evacuated tubes containing silica-coated film, Venoject II® clot activator film-containing tubes  as a variation of silica-coated tubes. The specifications of these tube brands are summarized in g for 14 min (A-PRF protocol) using a Duo centrifuge  at ambient temperature (20–22 °C).Fresh blood samples (approximately 9.0 mL) from each donor were collected into two brands of evacuated silica-coated plastic tubes, VacuetteQuality checks were carried out on individual blood samples by performing platelet and other blood cell counts using a pocH 100iV automated hematology analyzer .Disinfectant, 70% ethanol (2 mL) was added to each tube which were then tapped several times and sonicated for 60 s in an ultrasonic cleaner . The suspension of detached silica microparticles was transferred into plastic dishes and air-dried on a clean bench.Dried silica microparticles were attached to carbon tape and directly examined under a scanning electron microscope (SEM)  with an accelerating voltage of 15 kV ,13.Pure water (2 mL) was added to each tube and silica microparticles were detached thoroughly as described above. Then, the concentrations of silica suspensions (100 μL) were determined using a spectrophotometer . In a preliminary study, we confirmed that KOH did not significantly influence either the silica microparticles or the spectrophotometric determination.A-PRF clots were gently compressed with a compression device  , cut intt-test . Differences with p values < 0.05 were considered significant.The data were expressed as mean ± standard deviation (SD). For two-group comparisons, statistical analyses were conducted to compare the mean values by Student The macroscopic appearance of the silica-coated plastic tube (Neotube) pre- and post-centrifugation is shown in The components on the hazy wall of pre-centrifugation tubes were examined using SEM. Microscopic observations of silica microparticles contained in Neotube, Vacuette and Venoject II tubes are shown in A-PRF-like clots formed in individual plastic tubes after centrifugation and the balance of A-PRF-like matrices and red thrombi are shown in SEM observations of silica microparticles embedded in and attached to individual A-PRF-like matrices are shown in The contents and percentages of silica microparticles contained in A-PRF-like matrices versus whole contents of pre-use tubes are shown in According to the manufacturers’ information , the silThe specific gravity of amorphous silica (2.15–2.30) is much higher than red blood cells (approximately 1.10); therefore, silica microparticles can be precipitated immediately after centrifugation under citrated conditions ,21. HoweDespite contamination of silica microparticles in the resulting A-PRF-like matrices, the amounts varied with tube brands. The surface area of the silica coating film is substantially smaller than those on the inner wall of the tube. Thus, it is obvious that the mass of the silica microparticles in Venoject II tubes is much lower than that in other tube brands (Vacuette and Neotube) and consequently the mass of silica microparticles included in A-PRF-like matrix in Venoject II tubes must be lower than those of other brand tubes. In addition to the mass of silica microparticles, the silica entrapment and retention processes are primarily thought to be influenced by other factors, such as appearance and the size of the microparticles. Furthermore, these factors may influence the recovery during extraction and light transparency during spectrophotometric determination. It is plausible that as the microparticles become smaller, the entrapment and retention become more difficult. Therefore, we think that the reduction in recovery may not be necessarily related to or caused by the reduction in entrapment and/or retention.On the other hand, even though amorphous silica is less toxic than crystalline silica and is actually used in a variety of products, including food and toothpaste, recent investigations have raised the possibility that amorphous silica can cause inflammation or cytotoxicity ,24,25,26In Japan, a new regulatory framework for regenerative medicine, including PRP/PRF therapy, was established in 2014, and individual hospitals and clinics are required to pass inspection by the regulatory agencies prior to treatment of their patients with PRP/PRF . NonetheThe major advantages of PRF are low cost and high safety. Introducing possible silica hazards to this balance seems risky and may hamper the advances in PRF therapy. O’Connell forthrightly raised his concern over its use in PRF preparation in his article  and his The most appropriate therapeutic methods are generally selected based on the balance between risk and benefit. PRF therapy alone is superior to other therapies using medicines or bone substitutes in terms of cost and safety. Thus, it can be confirmed that PRF therapy is advantageous based on the balance between low risk and low benefit. Until the possible health hazards of amorphous silica microparticles contaminated in PRF matrices cannot be ruled out by strong evidence, PRF therapy using silica-containing tubes has no convincing advantages over other costly therapies and only increases the risk of complications.