Specific Sequences Research Articles

A mito-y conundrum: is more general or less but specific sequence data optimal? A comparison of the ForenSeq mtDNA whole genome and control region kits

ABSTRACT Mitochondrial DNA (mtDNA) is more prevalent than genomic DNA, making it useful for challenging forensic samples such as degraded bones and hair shafts. A comparison of the ForenSeq whole genome and control region mtDNA kits was undertaken on bones burnt to high temperatures and hair shafts covering a range of hair colours and treatments. Haplogroup predictions were made from the same haplogroup branch per donor for one bone burnt ≥ 700°C and all hair segments. Overall, the whole genome mtDNA kit was both more successful and produced more discriminatory haplogroup predictions than the control region kit.

Selective targeting of mu opioid receptors to primary cilia

Opioid receptors are therapeutically important G protein-coupled receptors (GPCRs) with diverse neuromodulatory effects. The functional consequences of opioid receptor activation are known to depend on receptor location in the plasma membrane, but mechanisms mediating selective localization of receptors to any particular membrane domain remain elusive. Here, we demonstrate the targeting of the mu opioid receptor (MOR) to the primary cilium, a discrete microdomain of the somatic plasma membrane, both invivo and in cultured cells. We further show that ciliary targeting is specific to MORs, requires a 17-residue sequence unique to the MOR cytoplasmic tail, and additionally requires the Tubby-like protein 3 (TULP3) ciliary adaptor protein. Our results reveal the potential for opioid receptors to undergo selective localization to the primary cilium. We propose that ciliary targeting is mediated through an elaboration of the recycling pathway, directed by a specific C-terminal recycling sequence in cis and requiring TULP3 in trans.

An aldehyde dehydrogenase gene, GhALDH7B4_A06, positively regulates fiber strength in upland cotton (Gossypium hirsutum L.).

High fiber strength (FS) premium cotton has significant market demand. Consequently, enhancing FS is a major objective in breeding quality cotton. However, there is a notable lack of known functionally applicable genes that can be targeted for breeding. To address this issue, our study used specific length-amplified fragment sequencing combined with bulk segregant analysis to study FS trait in an F2 population. Subsequently, we integrated these results with previous quantitative trait locus mapping results regarding fiber quality, which used simple sequence repeat markers in F2, F2:3, and recombinant inbred line populations. We identified a stable quantitative trait locus qFSA06 associated with FS located on chromosome A06 (90.74-90.83 Mb). Within this interval, we cloned a gene, GhALDH7B4_A06, which harbored a critical mutation site in coding sequences that is distinct in the two parents of the tested cotton line. In the paternal parent Ji228, the gene is normal and referred to as GhALDH7B4_A06O; however, there is a nonsense mutation in the maternal parent Ji567 that results in premature termination of protein translation, and this gene is designated as truncated GhALDH7B4_A06S. Validation using recombinant inbred lines and gene expression analysis revealed that this mutation site is correlated with cotton FS. Virus-induced gene silencing of GhALDH7B4 in cotton caused significant decreases in FS and fiber micronaire. Conversely, GhALDH7B4_A06O overexpression in Arabidopsis boosted cell wall component contents in the stem. The findings of our study provide a candidate gene for improving cotton fiber quality through molecular breeding.

Metathesis in Language Acquisition of Greek

This study investigates metathesis in children so that to see if it facilitates their language development and in which way. For this reason, thirteen Greek-speaking children are examined varying in age from 2;6.9 to 6;1.26. The data come from picture naming and spontaneous speech. Their examination reveals that its emergence lies to segmental, prosodic and phonotactic reasons. More specifically, metathesis helps children avoid specific sequences of consonants by switching them positions. It also assists them to avoid clusters located in unstressed syllables or illicit structures in the ambient language, as complex codas in loanwords. All metathesized segments are located in positions that can occupy according to the rules of Greek. For the data analysis, Optimality Theory is used (Prince & Smolensky, 1993), while we adopt the Multiple Parallel Grammars model (Revithiadou & Tzakosta, 2004) for the two different patterns attested in their speech.

P1' specificity of the S219V/R203G mutant tobacco etch virus protease.

Proteases that recognize linear amino acid sequences with high specificity became indispensable tools of recombinant protein technology for the removal of various fusion tags. Due to its stringent sequence specificity, the catalytic domain of the nuclear inclusion cysteine protease of tobacco etch virus (TEV PR) is also a widely applied reagent for enzymatic removal of fusion tags. For this reason, efforts have been made to improve its stability and modify its specificity. For example, P1' autoproteolytic cleavage-resistant mutant (S219V) TEV PR was found not only to be nearly impervious to self-inactivation, but also exhibited greater stability and catalytic efficiency than the wild-type enzyme. An R203G substitution has been reported to further relax the P1' specificity of the enzyme, however, these results were obtained from crude intracellular assays. Until now, there has been no rigorous comparison of the P1' specificity of the S219V and S219V/R203G mutants in vitro, under carefully controlled conditions. Here, we compare the P1' amino acid preferences of these single and double TEV PR mutants. The in vitro analysis was performed by using recombinant protein substrates representing 20 P1' variants of the consensus TENLYFQ*SGT cleavage site, and synthetic oligopeptide substrates were also applied to study a limited set of the most preferred variants. In addition, the enzyme-substrate interactions were analyzed in silico. The results indicate highly similar P1' preferences for both enzymes, many side-chains can be accommodated by the S1' binding sites, but the kinetic assays revealed lower catalytic efficiency for the S219V/R203G than for the S219V mutant.

Unexpected tobacco etch virus (TEV) protease cleavage of recombinant human proteins

The tobacco etch virus (TEV) protease is a commonly used reagent for removal of solubility and purification tags from recombinant proteins and is cited as being highly specific for its canonical cleavage site. Flexibility in some amino acids within this recognition sequence has been described in the literature but researchers generally assume few native human proteins will carry off-target sequences for TEV cleavage. We report here the aberrant cleavage of three human proteins with non-canonical TEV protease cleavage sites and identify broader sequence specificity rules that can be used to predict unwanted cleavage of recombinant proteins. Using these rules, 456 human proteins were identified that could be substrates for unwanted TEV protease cleavage.

Low-cost inkjet-printed nanostructured biosensor based on CRISPR/Cas12a system for pathogen detection

The escalating global incidence of infectious diseases caused by pathogenic bacteria, especially in developing countries, emphasises the urgent need for rapid and portable pathogen detection devices. This study introduces a sensitive and specific electrochemical biosensing platform utilising cost-effective electrodes fabricated by inkjet-printing gold and silver nanoparticles on a plastic substrate. The biosensor exploits the CRISPR/Cas12a system for detecting a specific DNA sequence selected from the genome of the target pathogen. Upon detection, the trans-activity of Cas12a/gRNA is triggered, leading to the cleavage of rationally designed single-strand DNA reporters (linear and hairpin) labelled with methylene blue (ssDNA-MB) and bound to the electrode surface. In principle, this sensing mechanism can be adapted to any bacterium by choosing a proper guide RNA to target a specific sequence of its DNA. The biosensor's performance was assessed for two representative pathogens (a Gram-negative, Escherichia coli, and a Gram-positive, Staphylococcus aureus), and results obtained with inkjet-printed gold electrodes were compared with those obtained by commercial screen-printed gold electrodes. Our results show that the use of inkjet-printed nanostructured gold electrodes, which provide a large surface area, in combination with the use of hairpin reporters containing a poly-T loop can increase the sensitivity of the assay corresponding to a signal variation of 86%. DNA targets amplified from various clinically isolated bacteria, have been tested and demonstrate the potential of the proposed platform for point-of-need applications.

Enzyme-driven converter and amplifier for inert double-stranded DNA without sequence restrictions

DNA molecules in life play a crucial role in various fields. However, DNA-based technologies confront significant difficulties in dealing with inert double-stranded DNA (dsDNA) with a blunt end without specific sequences. In this work, we utilized Thermus thermophilus Argonaute (TtAgo) and Vent DNA polymerase to construct an enzyme-driven device to convert and amplify arbitrary blunt-ended dsDNA into single-stranded DNA (ssDNA), thereby adapting to subsequent functional units. Based on the excellent performance of the dsDNA to ssDNA converter and amplifier (DSCA), inert dsDNA could be effectively identified. Additionally, DSCA accomplishes information interception and transmission, addressing the problem of dsDNA input response in DNA circuits and clinical diagnosis. Given these facts, this DSCA is expected to serve as a broad toolbox for the information transformation and amplification of inert dsDNA, prospering DNA circuit operations, and biosensing.

Precise editing of pathogenic nucleotide repeat expansions in iPSCs using paired prime editor.

Nucleotide repeat expansion disorders, a group of genetic diseases characterized by the expansion of specific DNA sequences, pose significant challenges to treatment and therapy development. Here, we present a precise and programmable method called prime editor-mediated correction of nucleotide repeat expansion (PE-CORE) for correcting pathogenic nucleotide repeat expansion. PE-CORE leverages a prime editor and paired pegRNAs to achieve targeted correction of repeat sequences. We demonstrate the effectiveness of PE-CORE in HEK293T cells and patient-derived induced pluripotent stem cells (iPSCs). Specifically, we focus on spinal and bulbar muscular atrophy and spinocerebellar ataxia type, two diseases associated with nucleotide repeat expansion. Our results demonstrate the successful correction of pathogenic expansions in iPSCs and subsequent differentiation into motor neurons. Specifically, we detect distinct downshifts in the size of both the mRNA and protein, confirming the functional correction of the iPSC-derived motor neurons. These findings highlight PE-CORE as a precision tool for addressing the intricate challenges of nucleotide repeat expansion disorders, paving the way for targeted therapies and potential clinical applications.

Separating spatial representations from polarity encoding in the processing of number and sequence stimuli in a four-way classification task

Although the SNARC effect in the processing of most magnitude stimuli and sequence stimuli has been reported for the past 30 years, it remains unclear whether this effect is caused by the spatial representation or polarity encoding of stimuli. In the present study, we designed five experiments using a four-way classification task to evaluate the ability of spatial representation theory and polarity encoding theory to explain the SNARC effect in the processing of number and sequence stimuli. In all five experiments in the present study, stimuli (Experiments 1 and 4: four different Arabic numbers, Experiment 2: sequence stimuli, Experiment 3: ordinal sequences relevant to working memory, Experiment 5: Chinese characters without any implicit spatial information) were centrally presented. Participants were asked to respond to specific number or sequence stimuli by pressing the A, S, K, and L keys in consistent trials (or the L, K, S, and A keys in inconsistent trials). The results showed that (1) the SNARC effect occurred in the processing of number and sequence stimuli both when only one specific number was mapped to one specific key (Experiments 1, 2 and 3) and when two numbers were mapped to one specific key (Experiment 4). (2) There was not a SNARC effect when the numbers were replaced with Chinese characters without any implicit spatial information (Experiment 5). The results of these five experiments imply that the SNARC effect in the processing of magnitude stimuli, including numbers and sequences, originates from the spatial representation of stimuli, supporting spatial representation theory.

Differential Solvent DEEP-STD NMR and MD Simulations Enable the Determinants of the Molecular Recognition of Heparin Oligosaccharides by Antithrombin to Be Disentangled.

The interaction of heparin with antithrombin (AT) involves a specific sequence corresponding to the pentasaccharide GlcNAc/NS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S (AGA*IA). Recent studies have revealed that two AGA*IA-containing hexasaccharides, which differ in the sulfation degree of the iduronic acid unit, exhibit similar binding to AT, albeit with different affinities. However, the lack of experimental data concerning the molecular contacts between these ligands and the amino acids within the protein-binding site prevents a detailed description of the complexes. Differential epitope mapping (DEEP)-STD NMR, in combination with MD simulations, enables the experimental observation and comparison of two heparin pentasaccharides interacting with AT, revealing slightly different bound orientations and distinct affinities of both glycans for AT. We demonstrate the effectiveness of the differential solvent DEEP-STD NMR approach in determining the presence of polar residues in the recognition sites of glycosaminoglycan-binding proteins.

Development and testing of a versatile genome editing application reporter (V-GEAR) system

CRISPR/Cas9 and novel cas fusion proteins leveraging specific DNA targeting ability combined with deaminases or reverse transcriptases has revolutionized genome editing. However, their efficacy heavily relies upon protein variants, targeting sgRNAs, and surrounding DNA sequence context within the targeted loci. This necessitates the need for efficient and rapid screening methods to evaluate these editing reagents and designs. Existing plasmid-based reporters lack flexibility, being fixed to specific DNA sequences, hindering direct comparisons between various editing approaches. To address this, we developed the versatile genome editing application reporter (V-GEAR) system. V-GEAR comprises genes detectable after desired editing via base editing, prime editing, or homology-directed repair within relevant genomic contexts. It employs a detectable synthetic cell surface protein (RQR8) followed by a customizable target sequence resembling genomic regions of interest. These genes allow for reliable identification of corrective editing and cell enrichment. We validated the V-GEAR system with base editors, prime editors, and Cas9 mediated homology-directed repair. Furthermore, the V-GEAR system offers versatility by allowing transient screening or stable integration at the AAVS1 safe harbor loci, rapidly achieved through immunomagnetic isolation. This innovative system enables direct comparisons among editing technologies, accelerating the development and testing of genome editing approaches.

HYPK: A marginally disordered protein sensitive to charge decoration

Intrinsically disordered proteins (IDPs) that lie close to the empirical boundary separating IDPs and folded proteins in Uversky's charge-hydropathy plot may behave as "marginal IDPs" and sensitively switch conformation upon changes in environment (temperature, crowding, and charge screening), sequence, or both. In our search for such a marginal IDP, we selected Huntingtin-interacting protein K (HYPK) near that boundary as a candidate; PKIα, also near that boundary, has lower secondary structure propensity; and Crk1, just across the boundary on the folded side, has higher secondary structure propensity. We used a qualitative Förster resonance energy transfer-based assay together with circular dichroism to simultaneously probe global and local conformation. HYPK shows several unique features indicating marginality: a cooperative transition in end-to-end distance with temperature, like Crk1 and folded proteins, but unlike PKIα; enhanced secondary structure upon crowding, in contrast to Crk1 and PKIα; and a cross-over from salt-induced expansion to compaction at high temperature, likely due to a structure-to-disorder transition not seen in Crk1 and PKIα. We then tested HYPK's sensitivity to charge patterning by designing charge-flipped variants including two specific sequences with identical amino acid composition that markedly differ in their predicted size and response to salt. The experimentally observed trends, also including mutants of PKIα, verify the predictions from sequence charge decoration metrics. Marginal proteins like HYPK show features of both folded and disordered proteins that make them sensitive to physicochemical perturbations and structural control by charge patterning.

Neuroinflammation and the role of epigenetic-based therapies for Huntington's disease management: the new paradigm.

Huntington's disease (HD) is an inherited, autosomal, neurodegenerative ailment that affects the striatum of the brain. Despite its debilitating effect on its patients, there is no proven cure for HD management as of yet. Neuroinflammation, excitotoxicity, and environmental factors have been reported to influence the regulation of gene expression by modifying epigenetic mechanisms. Aside focusing on the etiology, changes in epigenetic mechanisms have become a crucial factor influencing the interaction between HTT protein and epigenetically transcribed genes involved in neuroinflammation and HD. This review presents relevant literature on epigenetics with special emphasis on neuroinflammation and HD. It summarizes pertinent research on the role of neuroinflammation and post-translational modifications of chromatin, including DNA methylation, histone modification, and miRNAs. To achieve this about 1500 articles were reviewed via databases like PubMed, ScienceDirect, Google Scholar, and Web of Science. They were reduced to 534 using MeSH words like 'epigenetics, neuroinflammation, and HD' coupled with Boolean operators. Results indicated that major contributing factors to the development of HD such as mitochondrial dysfunction, excitotoxicity, neuroinflammation, and apoptosis are affected by epigenetic alterations. However, the association between neuroinflammation-altered epigenetics and the reported transcriptional changes in HD is unknown. Also, the link between epigenetically dysregulated genomic regions and specific DNA sequences suggests the likelihood that transcription factors, chromatin-remodeling proteins, and enzymes that affect gene expression are all disrupted simultaneously. Hence, therapies that target pathogenic pathways in HD, including neuroinflammation, transcriptional dysregulation, triplet instability, vesicle trafficking dysfunction, and protein degradation, need to be developed.

Enzyme-Inspired Ligand Engineering of Gold Nanoclusters for Electrocatalytic Microenvironment Manipulation.

Natural enzymes intricately regulate substrate accessibility through specific amino acid sequences and folded structures at their active sites. Achieving such precise control over the microenvironment has proven to be challenging in nanocatalysis, especially in the realm of ligand-stabilized metal nanoparticles. Here, we use atomically precise metal nanoclusters (NCs) as model catalysts to demonstrate an effective ligand engineering strategy to control the local concentration of CO2 on the surface of gold (Au) NCs during electrocatalytic CO2 reduction reactions (CO2RR). The precise incorporation of two 2-thiouracil-5-carboxylic acid (TCA) ligands within the pocket-like cavity of [Au25(pMBA)18]- NCs (pMBA = para-mercaptobenzoic acid) leads to a substantial acceleration in the reaction kinetics of CO2RR. This enhancement is attributed to a more favorable microenvironment in proximity to the active site for CO2, facilitated by supramolecular interactions between the nucleophilic Nδ- of the pyrimidine ring of the TCA ligand and the electrophilic Cδ+ of CO2. A comprehensive investigation employing absorption spectroscopy, mass spectrometry, isotopic labeling measurements, electrochemical analyses, and quantum chemical computation highlights the pivotal role of local CO2 enrichment in enhancing the activity and selectivity of TCA-modified Au25 NCs for CO2RR. Notably, a high Faradaic efficiency of 98.6% toward CO has been achieved. The surface engineering approach and catalytic fundamentals elucidated in this study provide a systematic foundation for the molecular-level design of metal-based electrocatalysts.

Effect of temperature on high strain-rate damage evolving in CFRP studied by synchrotron-based MHz X-ray phase contrast imaging

The present study demonstrates experimental evidence of subsurface mesoscale damage initiation and evolution in angle-ply CFRP laminates under high strain-rate loading at low temperatures using synchrotron-based X-ray MHz radiography. A bespoke set of loading, temperature control and in-situ X-ray imaging systems were applied to simultaneously correlate high strain-rate mechanical response with observed subsurface damage in a time-resolved manner. The results demonstrate that independent of temperature, damage evolved following a specific sequence; firstly intra-ply shear cracking along the fibre direction, developing into multi-layer cracking with continued deformation, and finally culminating in inter-ply delamination and complete failure of the specimen. The timescale for this sequence, however, was observed to strongly depend upon temperature, with low temperatures resulting in more rapid damage evolution and loss of mechanical strength.

Protein characteristics substantially influence the propensity of activity cliffs among kinase inhibitors

Activity cliffs (ACs) are pairs of structurally similar molecules with significantly different affinities for a biotarget, posing a challenge in computer-assisted drug discovery. This study focuses on protein kinases, significant therapeutic targets, with some exhibiting ACs while others do not despite numerous inhibitors. The hypothesis that the presence of ACs is dependent on the target protein and its complete structural context is explored. Machine learning models were developed to link protein properties to ACs, revealing specific tripeptide sequences and overall protein properties as critical factors in ACs occurrence. The study highlights the importance of considering the entire protein matrix rather than just the binding site in understanding ACs. This research provides valuable insights for drug discovery and design, paving the way for addressing ACs-related challenges in modern computational approaches.

Decentralized deadlock-free enforcement of message orderings in message-based systems

Message-based systems usually consist of distributed components that communicate using asynchronous message passing. In such systems, particular message orderings may violate some required properties. Given an automata-based specification of unwanted message sequences, we propose a decentralized deadlock-free runtime enforcement algorithm to prevent the formation of such sequences. In our approach, components are equipped with monitors executed concurrently. A component is only blocked before sending or receiving the last message of a sequence, until its associated monitor checks that such a message does not complete an unwanted sequence. According to the specification of unwanted sequences, some blocked components may suffer from a deadlock. Our deadlock-free algorithm guarantees that monitors detect and resolve such deadlocks by improving the existing deadlock detection algorithms. We evaluate the efficiency and scalability of our approach in terms of the communication overhead, the prevention latency, and the overhead of deadlock detection through simulation.

Cold atmospheric pressure plasma assisted rapid assembly of peptide-based structures: a molecular scaffold to form supramolecular architectures

The use of cold atmospheric pressure plasma (CAPP) technology in the production of peptide-based materials has shown great potential in modern technology. Herein, two aggregation-prone oligopeptides, GNNQQNY and KLVFFA, were subjected to CAPP treatment to form supramolecular assemblies/aggregates. Through peptide engineering and biophysical techniques, the effect of CAPP-generated reactive oxygen and nitrogen species on the oligopeptides were investigated for different treatment times revealing that the formation of these aggregates were primarily driven by electrostatic interactions without any chemical modifications. Field emission-scanning electron microscopy and Thioflavin T (ThT) binding assay confirmed the presence of distinct β-strands, particularly in the aggregates of the KLVFFA peptide upon CAPP irradiation. The combination of CAPP technology with peptide self-assembly process and the characterization techniques employed in this study holds promise for the development of such peptide supramolecular structures based on the specific amino acid sequences.

A lateral flow assay for miRNA-21 based on CRISPR/Cas13a and MnO2 nanosheets-mediated recognition and signal amplification.

The point-of-care testing (POCT) of miRNA has significant application in medical diagnosis, yet presents challenges due to their characteristics of high homology, low abundance, and short length, which hinders the achievement of quick detection with high specificity and sensitivity. In this study, a lateral flow assay based on the CRISPR/Cas13a system and MnO2 nanozyme was developed for highlysensitive detection of microRNA-21 (miR-21). The CRISPR/Cas13a cleavage system exhibits the ability to recognize the specific oligonucleotide sequence, where two-base mismatches significantly impact the cleavage activity of the Cas13a. Upon binding of the target to crRNA, the cleavage activity of Cas13a is activated, resulting in the unlocking of the sequence and initiating strand displacement, thereby enabling signal amplification to produce a new sequence P1. When applying the reaction solution to the lateral flow test strip, P1 mediates the capture of MnO2 nanosheets (MnO2 NSs) on the T zone, which catalyzes the oxidation of the pre-immobilized colorless substrate 3,3',5,5'-tetramethylbenzidine (TMB) on the T zone and generates the blue-green product (ox-TMB). The change in gray value is directly proportional to the concentration of miR-21, allowing for qualitative detection through visual inspection and quantitative measurement using ImageJ software. This method achieves the detection of miR-21 within a rapid 10-min timeframe, and the limit of detection (LOD) is 0.33pM. With the advantages of high specificity, simplicity, and sensitivity, the lateral flow test strip and the design strategy hold great potential for the early diagnosis of related diseases.