Fragment Length Research Articles

Head removal enhances planarian electrotaxis.

ABSTRACTCertain animal species utilize electric fields for communication, hunting and spatial orientation. Freshwater planarians move toward the cathode in a static electric field (cathodic electrotaxis). This planarian behavior was first described by Raymond Pearl more than a century ago. However, planarian electrotaxis has received little attention since, and the underlying mechanisms and evolutionary significance remain unknown. To close this knowledge gap, we developed an apparatus and scoring metrics for automated quantitative and mechanistic studies of planarian behavior upon exposure to a static electric field. Using this automated setup, we characterized electrotaxis in the planarian Dugesia japonica and found that this species responds to voltage instead of current, in contrast to results from previous studies using other planarian species. Surprisingly, we found differences in electrotaxis ability between small (shorter) and large (longer) planarians. To determine the cause of these differences, we took advantage of the regenerative abilities of planarians and compared electrotaxis in head, tail and trunk fragments of various lengths. We found that tail and trunk fragments electrotaxed, whereas head fragments did not, regardless of size. Based on these data, we hypothesized that signals from the head may interfere with electrotaxis when the head area/body area reached a critical threshold. In support of this hypothesis, we found that (1) smaller intact planarians that cannot electrotax have a relatively larger head-to-body-ratio than large planarians that can electrotax, and (2) the electrotaxis behavior of cut head fragments was negatively correlated with the head-to-body ratio of the fragments. Moreover, we could restore cathodic electrotaxis in head fragments via decapitation, directly demonstrating inhibition of electrotaxis by the head.

Identification of fusion transcripts in sarcoma from archival formalin-fixed paraffin-embedded tissues: A next-generation sequencing approach.

Most sarcomas are highly aggressive, and cause necrosis and hemorrhage. The diagnosis of sarcoma is challenging because of the lack of specificity of immunohistochemical staining; however, molecular biological approaches, such as genetic mutation, chromosomal translocation, and gene amplification, are promising. In this study, we extracted RNA from formalin-fixed paraffin-embedded (FFPE) tissue derived from surgically resected specimens of sarcoma stored for various periods and performed next-generation sequencing (NGS) analysis by MiniSeq using the Archer Fusion-Plex Sarcoma Panel. RNA was extracted from 63 FFPE tissue samples, and the degree of RNA degradation was assessed. The number of reads and fragment lengths were evaluated by NGS analysis. RNA extraction and cDNA synthesis were successful in 56 cases and library preparation was possible. Fusion genes were detected in 16 of 63 archived FFPE tissue samples in this study. However, in 18 cases, fragmentation was strong, and high-quality libraries could not be obtained. Nevertheless, comprehensive analysis of fusion genes with high sequence specificity by NGS can be a powerful alternative to reverse transcription-polymerase chain reaction and fluorescence in situ hybridization methods.

Pharmacogenetic profiling and individualised therapy in the treatment of degenerative spinal conditions.

Patients presenting with degenerative spinal changes are often poor surgical candidates due to associated co-morbidities, frailty, or sarcopenia. Additionally, surgeries of a degenerative spine can prove difficult due to the distortion of normal surgical anatomy. Therefore, many patients are managed conservatively with a variety of modalities, including over-the-counter and prescription medications. Nevertheless, several patients do not experience adequate relief from pain with analgesic medications, precipitating multiple hospital visits, and usage of resources. As a result, back pain is regarded as a major economic burden, with total costs of associated treatment exceeding $100 billion annually. Pharmacogenetics is a relatively novel method of evaluating an individual's response to analgesic medications, through analysis of germline polymorphisms. It entails obtaining a genetic sample, often via buccal swab or peripheral blood sample, and genetic analysis achieved through either polymerase chain reaction +/- Sanger sequencing, microassays, restriction length fragment polymorphism analysis, or genetic library preparation and next generation sequencing. The potential efficacy of pharmacogenetic analysis has been highlighted across several specialities to date. However, a paucity of evidence exists regarding spine surgery populations. Nevertheless, regular prospective pharmacogenetic analysis may ultimately prove beneficial when concerning degenerative spinal cohorts due to aforementioned surgical and economic considerations. The purpose of this narrative review is to outline how metaboliser profile variants affect the pharmacokinetics of specific analgesia used to treat back pain, and to discuss the current potential and limitations of employing regular pharmacogenetic analysis for spine surgery populations with degenerative conditions.

REViewer: haplotype-resolved visualization of read alignments in and around tandem repeats

BackgroundExpansions of short tandem repeats are the cause of many neurogenetic disorders including familial amyotrophic lateral sclerosis, Huntington disease, and many others. Multiple methods have been recently developed that can identify repeat expansions in whole genome or exome sequencing data. Despite the widely recognized need for visual assessment of variant calls in clinical settings, current computational tools lack the ability to produce such visualizations for repeat expansions. Expanded repeats are difficult to visualize because they correspond to large insertions relative to the reference genome and involve many misaligning and ambiguously aligning reads.ResultsWe implemented REViewer, a computational method for visualization of sequencing data in genomic regions containing long repeat expansions and FlipBook, a companion image viewer designed for manual curation of large collections of REViewer images. To generate a read pileup, REViewer reconstructs local haplotype sequences and distributes reads to these haplotypes in a way that is most consistent with the fragment lengths and evenness of read coverage. To create appropriate training materials for onboarding new users, we performed a concordance study involving 12 scientists involved in short tandem repeat research. We used the results of this study to create a user guide that describes the basic principles of using REViewer as well as a guide to the typical features of read pileups that correspond to low confidence repeat genotype calls. Additionally, we demonstrated that REViewer can be used to annotate clinically relevant repeat interruptions by comparing visual assessment results of 44 FMR1 repeat alleles with the results of triplet repeat primed PCR. For 38 of these alleles, the results of visual assessment were consistent with triplet repeat primed PCR.ConclusionsRead pileup plots generated by REViewer offer an intuitive way to visualize sequencing data in regions containing long repeat expansions. Laboratories can use REViewer and FlipBook to assess the quality of repeat genotype calls as well as to visually detect interruptions or other imperfections in the repeat sequence and the surrounding flanking regions. REViewer and FlipBook are available under open-source licenses at https://github.com/illumina/REViewer and https://github.com/broadinstitute/flipbook respectively.

Benchmarking ultra-high molecular weight DNA preservation methods for long-read and long-range sequencing.

BackgroundStudies in vertebrate genomics require sampling from a broad range of tissue types, taxa, and localities. Recent advancements in long-read and long-range genome sequencing have made it possible to produce high-quality chromosome-level genome assemblies for almost any organism. However, adequate tissue preservation for the requisite ultra-high molecular weight DNA (uHMW DNA) remains a major challenge. Here we present a comparative study of preservation methods for field and laboratory tissue sampling, across vertebrate classes and different tissue types.ResultsWe find that storage temperature was the strongest predictor of uHMW fragment lengths. While immediate flash-freezing remains the sample preservation gold standard, samples preserved in 95% EtOH or 20–25% DMSO-EDTA showed little fragment length degradation when stored at 4°C for 6 hours. Samples in 95% EtOH or 20–25% DMSO-EDTA kept at 4°C for 1 week after dissection still yielded adequate amounts of uHMW DNA for most applications. Tissue type was a significant predictor of total DNA yield but not fragment length. Preservation solution had a smaller but significant influence on both fragment length and DNA yield.ConclusionWe provide sample preservation guidelines that ensure sufficient DNA integrity and amount required for use with long-read and long-range sequencing technologies across vertebrates. Our best practices generated the uHMW DNA needed for the high-quality reference genomes for phase 1 of the Vertebrate Genomes Project, whose ultimate mission is to generate chromosome-level reference genome assemblies of all ∼70,000 extant vertebrate species.

Estimating bloodstain formation time by quantitative analysis of mtDNA degradation

The estimation of bloodstain formation time is still an unsolved problem in forensic science and lacks accurate quantitative methods. Whether DNA can be adopted to estimate bloodstain formation time is still controversial, and there is no study to confirm the potential of mtDNA markers. To address these issues, a triple quantification method based on the ratio of mtDNA fragments of different lengths of COⅠ (mitochondrially encoded cytochrome c oxidase Ⅰ) for estimating bloodstain formation time was established. A total of 152 samples (140 old samples, 12 fresh samples) were collected and tested, and the absolute copies of different-sized fragments of COⅠ (304 bp, 120 bp, 41 bp) in all samples were quantified by SYBR Green real-time qPCR. The natural logarithms of two copy number ratios (304 bp/41 bp, 120 bp/41 bp) of COⅠ in old samples were calculated, which were used as degradation indexes to evaluate the degradation degree of mtDNA. The 140 old human blood samples from 1 to 14 years of storage were accumulated from casework of forensic practice to establish the method of estimating bloodstain formation time and used to analyze the impact of gender factors on the two degradation indexes, and 10 animal samples and 2 fresh human samples were collected to verify the human specificity of the method. There was a high correlation between degradation indexes and bloodstain formation time (the absolute values of correlation coefficients of these two degradation indexes were 0.901 and 0.758 respectively). A method with triple quantification and dual indexes estimating bloodstain formation timewas successfully established, which was highly human-specific. There was no statistically significant difference in degradation indexes between different gender samples (P > 0.05). This study confirmed that mtDNA can be utilized to estimate bloodstain formation time, which provides a new solution to the forensic problem of estimating the time of bloodstain formation.

Pocillopora spp. growth analysis on restoration structures in an Eastern Tropical Pacific upwelling area.

Coral reefs in Culebra Bay (North Pacific of Costa Rica) are threatened by multiple anthropogenic disturbances including global warming, overfishing, eutrophication, and invasive species outbreaks. It is possible to assist their recovery by implementing ecological restoration techniques. This study used artificial hexagonal steel structures, called “spiders” to compare growth of Pocillopora spp. coral fragments of different sizes. Three initial fragment class sizes were used: 2, 5 and 8 cm, with each class size having 42 initial fragments. Changes in fragment length, width and area were measured monthly from January to December 2020. Results showed an overall survivorship of 70.21%, and no significant differences in survivorship and linear growth rate were detected between class sizes. The linear growth rates are 4.49 ± 1.19 cm year−1, 5.35 ± 1.48 cm year−1 and 3.25 ± 2.22 cm year−1 for the 2, 5 and 8 cm initial class sizes, respectively. Our results do not show significant differences in growth rates between the different initial fragment sizes. However, since small fragments (2 cm) present higher mortality during the first month, we recommend using larger fragments. In addition, coral fragments grew 48% more during the non-upwelling season, which may suggest that it might be more effective and safer to start the restoration efforts during this period.

The Butterfly Fragment in Wedge-Shaped Femoral Shaft Fracture: Comparison of Two Different Surgical Methods.

ObjectiveOur study compared the results of wedge‐shaped femoral shaft fracture following intramedullary (IM) nailing with or without fixation of the third fragment.MethodsWe retrospectively reviewed patients presenting with femoral shaft fracture with AO/OTA type 32‐B from 2011 to 2016. Patients were divided into two groups: closed reduction without touching the third fragment and open reduction with fixation of the third fragment. The fragment ratio, fragment length, nail size, dynamization or not, mRUST scores, union rate, and union time were compared between the two groups. Risk factors of non‐union were also investigated, including sex, age, fracture pattern, fracture location, dynamization, nail size, fragment ratio, fragment size, and postoperative fragment displacement.ResultsA total of 80 patients met inclusion criteria, 20 patients with wedge‐shaped shaft femoral fracture were managed with IM nailing and open reduction with fixation of the third fragment. Sixty patients were treated with IM nail without touching the third fragment. The union rate for the fixation and non‐fixation groups were 60.0% and 81.7%, respectively. The mean union time for the fixation group was 19 months vs 14 months for the non‐fixation group. Multi‐regression analysis showed larger nail size (odds ratio: 2.26) and fixation of the third fragment (odds ratio: 0.18) influenced fracture healing.ConclusionsFixation of the third fragment in wedge‐shaped shaft femoral fracture results in a longer union time and lower union rate. In the management of femoral fracture with a third fragment, a larger nail size is recommended and fixation should be performed in a closed manner. Fixation of the fragment may achieve better fracture reduction. However, disruption of the vasculature and surrounding structures may further result in nonunion of the fracture site.

Abstract 3405: Characterizing treatment response in head and neck cancer through viral ctDNA quantification and fragment length

Abstract Background: Head and neck cancer (HNC) is the sixth most common cancer type worldwide, and despite decreases in the rates of Human Papillomavirus (HPV)-negative HNC, an alarming increase in HPV+ HNC is occurring. Circulating tumor (ct)DNA isolated from liquid biopsy has been shown to have clinical utility as a biomarker in cancer, with ctDNA levels and fragment size used to monitor treatment response. Methods: The aim of this study was to determine the presence of ctDNA in liquid biopsy (plasma and saliva) of HPV+ HNC patients to non-invasively monitor treatment response and disease progression. Blood and saliva samples were collected from 45 HPV+ HNC patients and 14 HPV-negative controls at the McGill University Health Centre before and/or after surgical resection. Samples were analyzed using droplet digital PCR (ddPCR) with primers and probes for HPV16/HPV18, the most common subtypes of HPV in HNC, accounting for 97% of cases in North America. Analysis was performed for all patients with confirmed follow-up scans and/or pathology. ctDNA levels were correlated with disease course as determined through imaging (>6 months post-sampling) and pathological examination. Results: ctDNA was detectable in 21/23 (91%) patients prior to treatment and 8/36 (22%) post treatment. In the two patients who were not positive for ctDNA prior to treatment, tumor tissue was found to be negative for HPV DNA despite being p16-positive by immunohistochemistry. Patients showed shifts in ctDNA fragment length in longitudinal samples following treatment. All post-treatment patients with no detectable ctDNA had no signs of recurrence/residual disease on follow-up imaging. In contrast, all 8 patients who tested positive for ctDNA post-treatment showed signs of recurrence on follow-up imaging. In our cohort, 14 patients had matched pre- and post-treatment samples and all showed major reductions in ctDNA post treatment compared to pre-treatment, with 12/14 patients having a complete loss of detectable ctDNA. Concordance between the presence of ctDNA in saliva and plasma was found in 86% of patients. ctDNA was only detected in one analyte in 6 patients (3 in plasma only and 3 in saliva only). Conclusion: HPV ctDNA was successfully detected in saliva and blood of patients with HPV-positive HNC, with a sensitivity of 92% for ctDNA prior to treatment, and a sensitivity of 100% when adjusted for tumor HPV status as opposed to p16 staining. The inclusion of both plasma and saliva in analysis increases the sensitivity of assays, with the use of both analytes allowing for the detection of ctDNA in patients with low plasma DNA levels and for patients who did not have salivary ctDNA, potentially due to inability to produce sufficient saliva or tumor location. The presence of ctDNA correlated with treatment response, indicating the potential of HPV ctDNA for monitoring tumor progression in HPV-positive HNC patients. Citation Format: Sarah Tadhg Ferrier, Anthony Zeitouni, Nader Sadeghi, Thupten Tsering, Julia V. Burnier. Characterizing treatment response in head and neck cancer through viral ctDNA quantification and fragment length [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3405.

Permutation-based frame synchronization method for data transmission systems with short packets

This paper describes the research, development, and implementation of a frame synchronization method for data transmission systems with short packets, specifically those that use nonseparable factorial coding. The key concept of the method is using a permutation as a syncword. This permutation must satisfy the following condition: the minimum Hamming distance to all its circular shifts must be the maximum. The frame synchronization method employs correlation processing and majority processing of data fragments transmitted through the communication channel, where the fragment length is equal to the syncword length. As a result, the proposed frame synchronization method can realize frame synchronization in data transmission systems with adverse noise conditions. Theoretical and practical assessment of the number of accumulated fragments required to establish frame synchronization indicates that implementing the proposed method reduces the amount of received data, which subsequently reduces the time required to establish a connection between the transmitter and receiver. In addition, interleaving accumulated fragments further reduces the required number of received fragments.

Assessing putative bias in prediction of anti-microbial resistance from real-world genotyping data under explicit causal assumptions

Whole genome sequencing (WGS) is quickly becoming the customary means for identification of antimicrobial resistance (AMR) due to its ability to obtain high resolution information about the genes and mechanisms that are causing resistance and driving pathogen mobility. By contrast, traditional phenotypic (antibiogram) testing cannot easily elucidate such information. Yet development of AMR prediction tools from genotype-phenotype data can be biased, since sampling is non-randomized. Sample provenience, period of collection, and species representation can confound the association of genetic traits with AMR. Thus, prediction models can perform poorly on new data with sampling distribution shifts. In this work –under an explicit set of causal assumptions– we evaluate the effectiveness of propensity-based rebalancing and confounding adjustment on antibiotic resistance prediction using genotype-phenotype AMR data from the Pathosystems Resource Integration Center (PATRIC). We select bacterial genotypes (encoded as k-mer signatures, i.e., DNA fragments of length k), country, year, species, and AMR phenotypes for the tetracycline drug class, preparing test data with recent genomes coming from a single country. We test boosted logistic regression (BLR) and random forests (RF) with/without bias-handling. On 10,936 instances, we find evidence of species, location and year imbalance with respect to the AMR phenotype. The crude versus bias-adjusted change in effect of genetic signatures on AMR varies but only moderately (selecting the top 20,000 out of 40+ million k-mers). The area under the receiver operating characteristic (AUROC) of the RF (0.95) is comparable to that of BLR (0.94) on both out-of-bag samples from bootstrap and the external test (n = 1085), where AUROCs do not decrease. We observe a 1 %–5 % gain in AUROC with bias-handling compared to the sole use of genetic signatures. In conclusion, we recommend using causally-informed prediction methods for modeling real-world AMR data; however, traditional adjustment or propensity-based methods may not provide advantage in all use cases and further methodological development should be sought.

Does a Customized 3D Printing Plate Based on Virtual Reduction Facilitate the Restoration of Original Anatomy in Fractures?

The purpose of this study was to evaluate the restoration of original anatomy after fixation of sawbone fractures using case-specific 3D printing plates based on virtual reduction (VR). Three-dimensional models of 28 tibia sawbones with cortical marking holes were obtained. The sawbones were fractured at various locations of the shaft and 3D models were obtained. The fractured models were reduced virtually and customized non-locking metal plates that fit the reduced model were produced via 3D printing. The fractured sawbones were actually fixed to the customized plate with nonlocking screws and 3D models were generated. With the proximal fragments of the 3D models overlapped, the changes in length, 3D angulation, and rotation of the distal fragment were evaluated. Compared to the intact model (IN), the virtual reduction model (VR) and the actual fixation model (AF) showed no significant differences in length. Compared to the IN, the VR and the AF had mean 3D angulations of 0.39° and 0.64°, respectively. Compared to the IN model, the VR and the AF showed mean rotations of 0.89° and 1.51°, respectively. A customized plate based on VR facilitates the restoration of near-original anatomy in fractures of tibial sawbone shaft.

Development and growth of massive coral Lobophyllia hemprichii transplanted on Pramuka Island, Thousand Islands, Jakarta

The condition of coral reefs in Indonesia is declining, one of the efforts to restore the condition is to carry out coral transplants. The method used in this research is fragmentation transplantation. This study was conducted to determine the effect of fragmentation (cutting) on coral species Lobophyllia hemprichii from F1 parent transplants carried out on Pramuka Island, Seribu Islands Jakarta. There were two types of coral cutting treatment, namely L1 (wound 1) and L2 (wound 2), with 14 replications. This study showed that there was no effect on the number of cuttings on the survival (SR) and growth of the sub-massive coral Lobophyllia hemprichii (P>0.05). All coral fragments were alive until the end of the observation (SR=100%) and there was an increase in fragment length every month in 2 treatments. The increase in length over 6 months in corals with 1 wound was 10.17 mm (from 67.48 mm to 79.65 mm) and in corals with 2 wounds was 11.74 mm (from 79.01 mm to 90.75 mm). Coral growth rate was also not significantly different between the two treatments (P>0.05), with a range of 1.54-1.94 mm/month (wound 1) and 1.49-3.16 mm/month (wound 2). The growth rate increased after the corals recovered from wound healing (after 5 months of transplantation).

In vitro evaluation of cyclic fatigue resistance of thermally treated novel nickel-titanium rotary instruments

The objective of this study was to evaluate the cyclic fatigue resistance of novel NiTi files (subjected to heat treatment) using the in vitro model (artificial canal). Twelve ProDesign Logic instruments - PDL 25/.06 (Easy Equipamentos Odontológicos, Belo Horizonte, Brazil) and 12 Protaper Next X2 instruments - PTN (tip 25) were included in this research. The Static cyclic fatigue test was performed with a grooved stainless steel block simulating a canal of 1,5 mm diameters, a 60o angle of curvature and 5 mm radius. The files were positioned inside the artificial canal and rotated until the fracture occurred. Using the time until fracture (seconds) and the number of rotations per minute (RPM), the number of cycles until fracture (NCF) was calculated and the length of ruptured fragments was registered. Three samples from each group were analyzed in Scanning Electronic Microscopy (SEM) to characterize the metal rupture. Data were analyzed using the Mann-Whitney non-parametric test and the level of significance considered was p<0.05. PDL obtained a mean value of 956,0 NCF (352,0 - SD) and PTN achieved 391,0 NCF (36.7 - SD) with statistical significance between the groups (p < 0.001). There was no statistical difference on fragment length (p > 0.05). SEM analysis showed features compatible with ductile fracture. ProDesign LOGIC files showed higher NCF than Protaper Next X2. There was no difference between groups considering the length of the ruptured fragment.

Longer amplicons provide better sensitivity for electrochemical sensing of viral nucleic acid in water samples using PCB electrodes

The importance of monitoring environmental samples has gained a lot of prominence since the onset of COVID-19 pandemic, and several surveillance efforts are underway using gold standard, albeit expensive qPCR-based techniques. Electrochemical DNA biosensors could offer a potential cost-effective solution suitable for monitoring of environmental water samples in lower middle income countries. In this work, we demonstrate electrochemical detection of amplicons as long as {503},hbox {bp} obtained from Phi6 bacteriophage (a popular surrogate for SARS-CoV-2) isolated from spiked lake water samples, using ENIG finish PCB electrodes with no surface modification. The electrochemical sensor response is thoroughly characterised for two DNA fragments of different lengths ({117},hbox {bp} and {503},hbox {bp}), and the impact of salt in PCR master mix on methylene blue (MB)-DNA interactions is studied. Our findings establish that length of the DNA fragment significantly determines electrochemical sensitivity, and the ability to detect long amplicons without gel purification of PCR products demonstrated in this work bodes well for realisation of fully-automated solutions for in situ measurement of viral load in water samples.

Systematic review and meta-analysis: Water type and temperature affect environmental DNA decay.

Environmental DNA (eDNA) has been used in a variety of ecological studies and management applications. The rate at which eDNA decays has been widely studied but at present it is difficult to disentangle study‐specific effects from factors that universally affect eDNA degradation. To address this, a systematic review and meta‐analysis was conducted on aquatic eDNA studies. Analysis revealed eDNA decayed faster at higher temperatures and in marine environments (as opposed to freshwater). DNA type (mitochondrial or nuclear) and fragment length did not affect eDNA decay rate, although a preference for <200 bp sequences in the available literature means this relationship was not assessed with longer sequences (e.g. >800 bp). At present, factors such as ultraviolet light, pH, and microbial load lacked sufficient studies to feature in the meta‐analysis. Moving forward, we advocate researching these factors to further refine our understanding of eDNA decay in aquatic environments.

ANI analysis of poxvirus genomes reveals its potential application to viral species rank demarcation.

Average nucleotide identity (ANI) is a prominent approach for rapidly classifying archaea and bacteria by recruiting both whole genomic sequences and draft assemblies. To evaluate the feasibility of ANI in virus taxon demarcation, 685 poxviruses were assessed. Prior to the analysis, the fragment length and threshold of the ANI value were optimized as 200 bp and 98 per cent, respectively. After ANI analysis and network visualization, the resulting sixty-one species (ANI species rank) were clustered and largely consistent with the groupings found in National Center for Biotechnology Information Virus [within the International Committee on Taxonomy of Viruses (ICTV) Master Species List]. The species identities of thirty-four other poxviruses (excluded by the ICTV Master Species List) were also identified. Subsequent phylogenetic analysis and Guanine-Cytosine (GC) content comparison done were found to support the ANI analysis. Finally, the BLAST identity of concatenated sequences from previously identified core genes showed 91.8 per cent congruence with ANI analysis at the species rank, thus showing potential as a marker gene for poxviruses classification. Collectively, our results reveal that the ANI analysis may serve as a novel and efficient method for poxviruses demarcation.

LncRNAs imported into mitochondria possess distinct features stratified by machine learning that promote interaction with the mitochondrial import protein PNPase

Understanding the localization and regulatory activity of extra‐nuclear long non‐coding RNAs (lncRNAs) is especially critical in the context of the mitochondrion, which possesses a genome separate from the nucleus that dictates bioenergetic function and is influenced by a broad range of pathologies. Prior research including efforts from our own laboratory have identified Polynucleotide Phosphorylase (PNPase) to be critical for selective RNA passage through the mitochondrial membrane, including microRNAs and other ncRNAs. However, the dynamics of lncRNA binding with PNPase have not yet been determined. Understanding how lncRNAs interact with PNPase is a crucial initial step for predicting their ability to be imported into mitochondria and how the process can be manipulated to regulate mitochondrial genome expression.The objective of this study was to evaluate lncRNAs present in mitochondria and classify sequence‐based features that might permit interaction with PNPase. Sequencing performed on mitochondrial and cytoplasmic isolates from human and mouse cardiac tissue identified over 500 mitochondrially‐localized nuclear genome‐encoded lncRNAs. Crosslinked immunoprecipitation (CLIP) of mitochondrial isolates using antibodies for PNPase pulled down predominantly lncRNAs in both human and mouse. The most highly bound lncRNA sequences were run though supervised machine learning using 10‐fold cross validation through Support Vector Machines (SVM) and Classification and Regression Trees (CART) machine learning algorithms, which identified stratification of primary and secondary sequence features as compared to random RNA and low binding lncRNA sequences, with an accuracy of 82% for SVM and an area under curve value of 0.89 for CART. Primary sequence features such as dissimilarities from coding sequences, various k‐mer frequencies, and overall GC content correlated with increased lncRNA interaction with PNPase (p&lt;0.001), as did secondary features such as minimum free energy and electron‐ion interaction pseudopotential (p&lt;0.001). These features were integrated into an artificial RNA fragment sequence that was gel shifted with purified PNPase protein and showed higher binding affinity than a random RNA fragment of equal length.LncRNAs are a unique species of ncRNA with a diverse range of regulatory functions. Understanding the mechanism behind lncRNA import into the mitochondrion could lead to novel therapeutic interventions to preserve bioenergetic activity. This study indicates that PNPase has affinity for rankable primary sequence and secondary structural features within lncRNAs, and application of the most critical features can be used to improve interaction with PNPase and potentially import into mitochondria.

Engineering a single-chain variable fragment of cetuximab for CAR T-cell therapy against head and neck squamous cell carcinomas

The monoclonal antibody cetuximab recognizes domain III of the epithelial growth factor receptor (EGFR) with high-affinity and is an important element in the treatment of several malignancies that overexpress non-mutated wild-type EGFR. In order to create an EGFR recognizing chimeric antigen receptor (CAR) for cellular immunotherapy of head and neck squamous cell carcinoma (HNSCC), we rationally designed single chain fragments of different lengths based on the cetuximab variable heavy and light chains. We then cloned the different cetuximab fragments into our second generation CAR construct, expressed CARs on primary human T-cells from healthy donors using mono- or biscistronic lentiviral vectors and tested the stability, functionality and specificity of the CARs. Our smallest CAR construct was most efficient with greatly improved vector production and T-cell transduction efficacy. Finally, we demonstrated that the new cetuximab CAR construct expressed on T-cells is highly reactive against EGFR-positive HNSCCs and also malignant cells from other solid cancer entities. In conclusion, we generated an optimized high-affinity EGFR CAR construct for the next steps in cancer immunotherapy, which need to focus on the development of armored CAR T-cells that will be more resistant and effective in the hostile microenvironment present in solid cancers.

STRipy: A graphical application for enhanced genotyping of pathogenic short tandem repeats in sequencing data.

Expansions of short tandem repeats (STRs) have been implicated as the causal variant in over 50 diseases known to date. There are several tools which can genotype STRs from high‐throughput sequencing (HTS) data. However, running these tools out of the box only allows around half of the known disease‐causing loci to be genotyped. Furthermore, the genotypes estimated at these loci are often underestimated with maximum lengths limited to either the read or fragment length, which is less than the pathogenic cutoff for some diseases. Although analysis tools can be customized to genotype extra loci, this requires proficiency in bioinformatics to set up, limiting their widespread usage by other researchers and clinicians. To address these issues, we have developed a new software called STRipy, which is able to target all known disease‐causing STRs from HTS data. We created an intuitive graphical interface for STRipy and significantly simplified the detection of STRs expansions. Moreover, we genotyped all disease loci for over two and half thousand samples to provide population‐wide distributions to assist with interpretation of results. We believe the simplicity and breadth of STRipy will increase the genotyping of STRs in sequencing data resulting in further diagnoses of rare STR diseases.